Methods for screening for transdominant intracellular effector peptides and RNA molecules

ABSTRACT

Methods and compositions for screening for intracellular transdominant effector peptides and RNA molecules selected inside living cells from randomized pools are provided.

This is a continuation-in-part of U.S. application Ser. No. 08/589,108,filed Jan. 23, 1996, abandoned and U.S. application Ser. No. 08/589,911,filed Jan. 23, 1996, abandoned.

FIELD OF THE INVENTION

The technical field of this invention is methods for screening fortransdominant effector peptides and RNA molecules selected inside livingcells from randomized pools.

BACKGROUND OF THE INVENTION

Signaling pathways in cells often begin with an effector stimulus thatleads to a phenotypically describable change in cellular physiology.Despite the key role intracellular signaling pathways play in diseasepathogenesis, in most cases, little is understood about a signalingpathway other than the initial stimulus and the ultimate cellularresponse.

Historically, signal transduction has been analyzed by biochemistry orgenetics. The biochemical approach dissects a pathway in a"stepping-stone" fashion: find a molecule that acts at, or is involvedin, one end of the pathway, isolate assayable quantities and then try todetermine the next molecule in the pathway, either upstream ordownstream of the isolated one. The genetic approach is classically a"shot in the dark": induce or derive mutants in a signaling pathway andmap the locus by genetic crosses or complement the mutation with a cDNAlibrary. Limitations of biochemical approaches include a reliance on asignificant amount of pre-existing knowledge about the constituentsunder study and the need to carry such studies out in vitro,post-mortem. Limitations of purely genetic approaches include the needto first derive and then characterize the pathway before proceeding withidentifying and cloning the gene.

Screening molecular libraries of chemical compounds for drugs thatregulate signal systems has led to important discoveries of greatclinical significance. Cyclosporin A (CsA) and FK506, for examples, wereselected in standard pharmaceutical screens for inhibition of T-cellactivation. It is noteworthy that while these two drugs bind completelydifferent cellular proteins--cyclophilin and FK506 binding protein(FKBP), respectively, the effect of either drug is virtually thesame--profound and specific suppression of T-cell activation,phenotypically observable in T cells as inhibition of mRNA productiondependent on transcription factors such as NF-AT and NF-KB. Libraries ofsmall peptides have also been successfully screened in vitro in assaysfor bioactivity. The literature is replete with examples of smallpeptides capable of modulating a wide variety of signaling pathways. Forexample, a peptide derived from the HIV-1 envelope protein has beenshown to block the action of cellular calmodulin.

A major limitation of conventional in vitro screens is delivery. Whileonly minute amounts of an agent may be necessary to modulate aparticular cellular response, delivering such an amount to the requisitesubcellular location necessitates exposing the target cell or system torelatively massive concentrations of the agent. The effect of suchconcentrations may well mask or preclude the targeted response.

Thus, it is an object of the present invention to provide methods andcompositions for the effective introduction of random libraries intocells to screen for bioactive compounds.

Relevant Literature

Mann et al. (1983) Cell 33, 153-159, Pear et al. (1993) Proc. Natl.Acad. Sci. USA 90(18):8392-6 and WO 94/19478 describe the BOSC and BINGretroviral systems useful as delivery vectors for the disclosed methods.

Scott and Craig (1994) Current Opinion in Biotechnology 5:40-48 reviewrandom peptide libraries. Hupp et al. (1995) describe small peptideswhich activate the latent sequence-specific DNA binding function of p53.Palzkill et al. (1994) report the selection of functional signalcleavage sites from a library of random sequences introduced into TEM-1-lactamase.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for screening fortransdominant bioactive agents such as pharmaceuticals. The inventionaccesses molecules or targets within living cells and provides for thedirect selection of those bioactive agents with desired phenotypiceffects.

In one aspect of the invention, methods for screening for atransdominant bioactive agent capable of altering the phenotype of acell are provided. The methods comprise the steps of a) introducing amolecular library of randomized candidate nucleic acids into a pluralityof cells, wherein each of said nucleic acids comprises a differentnucleotide sequence; b) screening the plurality of cells for a cellexhibiting an altered phenotype, wherein the altered phenotype is due tothe presence of a transdominant bioactive agent. The methods may alsoinclude the steps of c) isolating the cell(s) exhibiting an alteredphenotype, d) isolating a candidate nucleic acid from the cell(s).

The invention further provides methods for isolating a target moleculeusing either a candidate nucleic acid or the expression product of acandidate nucleic acid.

In an additional aspect, the candidate nucleic acids of the inventionare linked to fusion partners.

In a further aspect, the invention provides methods for screening for atransdominant bioactive agent capable of altering the phenotype of acell. The methods comprises the steps of a) introducing a molecularlibrary of randomized candidate nucleic acids into a first plurality ofcells, wherein each of the nucleic acids comprises a differentnucleotide sequence; b) contacting the first plurality of cells with asecond plurality of cells; and c) screening the second plurality ofcells for a cell exhibiting an altered phenotype.

In an additional aspect, the present invention provides molecularlibraries of retroviruses comprising different randomized nucleic acids,and cellular libraries containing the retroviral libraries.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Creation of a library of random peptides in a retrovirus DNAconstruct by PCR.

FIG. 2. Creation of a library of random peptides in a retrovirus DNAconstruct by primed DNA synthesis. (SEQ ID NO:1); (SEQ ID NO:2); (SEQ IDNO:3).

FIG. 3. Presentation constructs for localizing presentation structuresto specific cellular locales.

FIG. 4. Schematic of a retroviral construct.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions to create,effectively introduce into cells and screen compounds that affect asignaling pathway. Little or no knowledge of the pathway is required,other than a presumed signaling event and an observable physiologicchange in the target cell. The disclosed methods are conceptuallydistinct from prior library search methods in that it is an in vivostratagem for accessing intracellular signaling mechanisms. Theinvention also provides for the isolation of the constituents of thepathway, the tools to characterize the pathway, and lead compounds forpharmaceutical development.

The present invention provides methods for the screening of candidatebioactive agents which are capable of altering the phenotype of cellscontaining the agents. The methods of the present invention provide asignificant improvement over conventional screening techniques, as theyallow the rapid screening of large numbers of random oligonucleotidesand their corresponding expression products in a single, in vivo step.Thus, by delivering the random oligonucleotides to cells and screeningthe same cells, without the need to collect or synthesize in vitro thecandidate agents, highly efficient screening is accomplished. Inaddition, the present methods allow screening in the absence ofsignificant prior characterization of the cellular defect per se.

Thus, the present invention provides methods for screening candidatebioactive agents for a transdominant bioactive agent capable of alteringthe phenotype of a cell.

By "candidate bioactive agents" or "candidate drugs" or "candidateexpression products" or grammatical equivalents herein is meant theexpression product of a candidate nucleic acid which may be tested forthe ability to transdominantly alter the phenotype of a cell. As isdescribed below, the candidate bioactive agents are the expressionproducts of candidate nucleic acids, and encompass several chemicalclasses, including peptides and nucleic acids such as DNA, messenger RNA(mRNA), antisense RNA, ribozyme components, etc. Thus, the candidatebioactive agents (expression products) may be either translationproducts of the candidate nucleic acids, i.e. peptides, or transcriptionproducts of the candidate nucleic acids, i.e. either DNA or RNA.

In a preferred embodiment, the candidate bioactive agents aretranslation products of the candidate nucleic acids. In this embodiment,the candidate nucleic acids are introduced into the cells, and the cellsexpress the nucleic acids to form peptides. Thus, in this embodiment,the candidate bioactive agents are peptides. Generally, peptides rangingfrom about 4 amino acids in length to about 100 amino acids may be used,with peptides ranging from about 5 to about 50 being preferred, withfrom about 5 to about 30 being particularly preferred and from about 6to about 20 being especially preferred.

In a preferred embodiment, the candidate bioactive agents aretranscription products of the candidate nucleic acids, and are thus alsonucleic acids. The transcription products may be either primarytranscripts or secondary translation products. That is, using theretroviral reverse transcriptase, primary DNA is made which are laterconverted into double stranded DNA. Additionally, using the primary DNA,RNA transcripts can be generated within the cell, including mRNA,antisense RNA and ribozymes or portions thereof.

At a minimum, the candidate bioactive agents comprise randomizedexpression products of the candidate nucleic acids. That is, everycandidate bioactive agent has a randomized portion, as defined below,that is the basis of the screening methods outlined herein. In addition,to the randomized portion, the candidate bioactive agent may alsoinclude a fusion partner.

In a preferred embodiment, the candidate bioactive agents are linked toa fusion partner. By "fusion partner" or "functional group" herein ismeant a sequence that is associated with the candidate bioactive agent,that confers upon all members of the library in that class a commonfunction or ability. Fusion partners can be heterologous (i.e. notnative to the host cell), or synthetic (not native to any cell).Suitable fusion partners include, but are not limited to: a)presentation structures, as defined below, which provide the candidatebioactive agents in a conformationally restricted or stable form; b)targeting sequences, defined below, which allow the localization of thecandidate bioactive agent into a subcellular or extracellularcompartment; c) rescue sequences as defined below, which allow thepurification or isolation of either the candidate bioactive agents orthe nucleic acids encoding them; d) stability sequences, which conferstability or protection from degradation to the candidate bioactiveagent or the nucleic acid encoding it, for example resistance toproteolytic degradation; e) dimerization sequences, to allow for peptidedimerization; or f) any combination of a), b), c), d), and e), as wellas linker sequences as needed.

In a preferred embodiment, the fusion partner is a presentationstructure. By "presentation structure" or grammatical equivalents hereinis meant a sequence, which, when fused to candidate bioactive agents,causes the candidate agents to assume a conformationally restrictedform. Proteins interact with each other largely through conformationallyconstrained domains. Although small peptides with freely rotating aminoand carboxyl termini can have potent functions as is known in the art,the conversion of such peptide structures into pharmacologic agents isdifficult due to the inability to predict side-chain positions forpeptidomimetic synthesis. Therefore the presentation of peptides inconformationally constrained structures will benefit both the latergeneration of pharmaceuticals and will also likely lead to higheraffinity interactions of the peptide with the target protein. This facthas been recognized in the combinatorial library generation systemsusing biologically generated short peptides in bacterial phage systems.A number of workers have constructed small domain molecules in which onemight present randomized peptide structures.

While the candidate bioactive agents may be either nucleic acid orpeptides, presentation structures are preferably used with peptidecandidate agents. Thus, synthetic presentation structures, i.e.artificial polypeptides, are capable of presenting a randomized peptideas a conformationally-restricted domain. Generally such presentationstructures comprise a first portion joined to the N-terminal end of therandomized peptide, and a second portion joined to the C-terminal end ofthe peptide; that is, the peptide is inserted into the presentationstructure, although variations may be made, as outlined below. Toincrease the functional isolation of the randomized expression product,the presentation structures are selected or designed to have minimalbiologically activity when expressed in the target cell.

Preferred presentation structures maximize accessibility to the peptideby presenting it on an exterior loop. Accordingly, suitable presentationstructures include, but are not limited to, minibody structures, loopson beta-sheet turns and coiled-coil stem structures in which residuesnot critical to structure are randomized, zinc-finger domains,cysteine-linked (disulfide) structures, transglutaminase linkedstructures, cyclic peptides, B-loop structures, helical barrels orbundles, leucine zipper motifs, etc.

In a preferred embodiment, the presentation structure is a coiled-coilstructure, allowing the presentation of the randomized peptide on anexterior loop. See, for example, Myszka et al., Biochem. 33:2362-2373(1994), hereby incorporated by reference, and FIG. 3). Using this systeminvestigators have isolated peptides capable of high affinityinteraction with the appropriate target. In general, coiled-coilstructures allow for between 6 to 20 randomized positions.

A preferred coiled-coil presentation structure is as follows:MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSKLAA CGPP (SEQ IDNO:4). The underlined regions represent a coiled-coil leucine zipperregion defined previously (see Martin et al., EMBO J. 13(22):5303-5309(1994), incorporated by reference). The bolded GRGDMP (SEQ ID NO:5)region represents the loop structure and when appropriately replacedwith randomized peptides (i.e. candidate bioactive agents, generallydepicted herein as (X)_(n), where X is an amino acid residue and n is aninteger of at least 5 (SEQ ID NO:98) or 6 (SEQ ID NO:80)) can be ofvariable length. The replacement of the bolded region is facilitated byencoding restriction endonuclease sites in the underlined regions, whichallows the direct incorporation of randomized oligonucleotides at thesepositions. For example, a preferred embodiment generates a XhoI site atthe double underlined LE site and a HindIII site at thedouble-underlined KL site.

In a preferred embodiment, the presentation structure is a minibodystructure. A "minibody" is essentially composed of a minimal antibodycomplementarity region. The minibody presentation structure generallyprovides two randomizing regions that in the folded protein arepresented along a single face of the tertiary structure. See for exampleBianchi et al., J. Mol. Biol. 236(2):649-59 (1994), and references citedtherein, all of which are incorporated by reference). Investigators haveshown this minimal domain is stable in solution and have used phageselection systems in combinatorial libraries to select minibodies withpeptide regions exhibiting high affinity, Kd=10⁻⁷, for thepro-inflammatory cytokine IL-6.

A preferred minibody presentation structure is as follows:MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTS QSILYLQKKKGPP(SEQ ID NO:6). The bold, underline regions are the regions which may berandomized. The italized phenylalanine must be invariant in the firstrandomizing region. The entire peptide is cloned in athree-oligonucleotide variation of the coiled-coil embodiment, thusallowing two different randomizing regions to be incorporatedsimultaneously. This embodiment utilizes non-palindromic BstXI sites onthe termini.

In a preferred embodiment, the presentation structure is a sequence thatcontains generally two cysteine residues, such that a disulfide bond maybe formed, resulting in a conformationally constrained sequence. Thisembodiment is particularly preferred when secretory targeting sequencesare used. As will be appreciated by those in the art, any number ofrandom sequences, with or without spacer or linking sequences, may beflanked with cysteine residues. In other embodiments, effectivepresentation structures may be generated by the random regionsthemselves. For example, the random regions may be "doped" with cysteineresidues which, under the appropriate redox conditions, may result inhighly crosslinked structured conformations, similar to a presentationstructure. Similarly, the randomization regions may be controlled tocontain a certain number of residues to confer β-sheet or α-helicalstructures.

In a preferred embodiment, the fusion partner is a targeting sequence.As will be appreciated by those in the art, the localization of proteinswithin a cell is a simple method for increasing effective concentrationand determining function. For example, RAF1 when localized to themitochondrial membrane can inhibit the anti-apoptotic effect of BCL-2.Similarly, membrane bound Sos induces Ras mediated signaling inT-lymphocytes. These mechanisms are thought to rely on the principle oflimiting the search space for ligands, that is to say, the localizationof a protein to the plasma membrane limits the search for its ligand tothat limited dimensional space near the membrane as opposed to the threedimensional space of the cytoplasm. Alternatively, the concentration ofa protein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. Finally, the ligand or target maysimply be localized to a specific compartment, and inhibitors must belocalized appropriately.

Thus, suitable targeting sequences include, but are not limited to,binding sequences capable of causing binding of the expression productto a predetermined molecule or class of molecules while retainingbioactivity of the expression product, (for example by using enzymeinhibitor or substrate sequences to target a class of relevant enzymes);sequences signalling selective degradation, of itself or co-boundproteins; and signal sequences capable of constitutively localizing thecandidate expression products to a predetermined cellular locale,including a) subcellular locations such as the Golgi, endoplasmicreticulum, nucleus, nucleoli, nuclear membrane, mitochondria,chloroplast, secretory vesicles, lysosome, and cellular membrane; and b)extracellular locations via a secretory signal. Particularly preferredis localization to either subcellular locations or to the outside of thecell via secretion.

In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val) (SEQ ID NO:7),Kalderon (1984), et al., Cell, 39:499-509; the human retinoic acidreceptor-β nuclear localization signal (ARRRRP) (SEQ ID NO:8); NFKB p50(EEVQRKRQKL (SEQ ID NO:9); Ghosh et al., Cell 62:1019 (1990); NFKB p65(EEKRKRTYE (SEQ ID NO:10); Nolan et al., Cell 64:961 (1991); and others(see for example Boulikas, J. Cell. Biochem. 55(1):32-58 (1994), herebyincorporated by reference) and double basic NLS's exemplified by that ofthe Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val LysArg Pro Ala Ala Thr Lys Lys Ala Gly Gin Ala Lys Lys Lys Lys Leu Asp)(SEQ ID NO:11), Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall,et al., J. Cell Biol., 107:641-849; 1988). Numerous localization studieshave demonstrated that NLSs incorporated in synthetic peptides orgrafted onto reporter proteins not normally targeted to the cell nucleuscause these peptides and reporter proteins to be concentrated in thenucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol.,2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci. USA,84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci. USA,87:458-462, 1990.

In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound peptide libraries are useful for both the identificationof important elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting therandomized expression product extracellularly or in the cytoplasmicspace; see FIG. 3. For extracellular presentation, a membrane anchoringregion is provided at the carboxyl terminus of the peptide presentationstructure. The randomized expression product region is expressed on thecell surface and presented to the extracellular space, such that it canbind to other surface molecules (affecting their function) or moleculespresent in the extracellular medium. The binding of such molecules couldconfer function on the cells expressing a peptide that binds themolecule. The cytoplasmic region could be neutral or could contain adomain that, when the extracellular randomized expression product regionis bound, confers a function on the cells (activation of a kinase,phosphatase, binding of other cellular components to effect function).Similarly, the randomized expression product-containing region could becontained within a cytoplasmic region, and the transmembrane region andextracellular region remain constant or have a defined function.

Membrane-anchoring sequences are well known in the art and are based onthe genetic geometry of mammalian transmembrane molecules. Peptides areinserted into the membrane based on a signal sequence (designated hereinas ssTM) and require a hydrophobic transmembrane domain (herein TM). Thetransmembrane proteins are inserted into the membrane such that theregions encoded 5' of the transmembrane domain are extracellular and thesequences 3' become intracellular. Of course, if these transmembranedomains are placed 5' of the variable region, they will serve to anchorit as an intracellular domain, which may be desirable in someembodiments. ssTMs and TMs are known for a wide variety of membranebound proteins, and these sequences may be used accordingly, either aspairs from a particular protein or with each component being taken froma different protein, or alternatively, the sequences may be synthetic,and derived entirely from consensus as artificial delivery domains.

As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

Useful sequences include sequences from: 1) class I integral membraneproteins such as IL-2 receptor beta-chain (residues 1-26 are the signalsequence, 241-265 are the transmembrane residues; see Hatakeyama et al.,Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem. 174:671(1988)) and insulin receptor beta chain (residues 1-27 are the signal,957-959 are the transmembrane domain and 960-1382 are the cytoplasmicdomain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 (1985)); 2)class II integral membrane proteins such as neutral endopeptidase(residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmicdomain; see Malfroy et al., Biochem. Biophys. Res. Commun. 144:59(1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5' end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP (SEQ ID NO:12);Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2(MSSFGYRTLTVALFTLICCPG (SEQ ID NO:13); Staunton et al., Nature (London)339:61 (1989)). These leader sequences deliver the construct to themembrane while the hydrophobic transmembrane domains, placed 3' of therandom candidate region, serve to anchor the construct in the membrane.These transmembrane domains are encompassed by amino acids 145-195 fromCD8 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR (SEQ ID

NO:14); Nakauchi, supra) and 224-256 from ICAM-2(MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR (SEQ ID NO:15); Staunton, supra).

Alternatively, membrane anchoring sequences include the GPI anchor,which results in a covalent bond between the molecule and the lipidbilayer via a glycosyl-phosphatidylinositol bond for example in DAF(PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:16), with the boldedserine the site of the anchor; see Homans et al., Nature333(6170):269-72 (1988), and Moran et al., J. Biol. Chem. 266:1250(1991)). In order to do this, the GPI sequence from Thy-1 can becassetted 3' of the variable region in place of a transmembranesequence.

Similarly, myristylation sequences can serve as membrane anchoringsequences. It is known that the myristylation of c-src recruits it tothe plasma membrane. This is a simple and effective method of membranelocalization, given that the first 14 amino acids of the protein aresolely responsible for this function: MGSSKSKPKDPSQR (SEQ ID NO:17) (seeCross et al., Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science262:1019-1024 (1993), both of which are hereby incorporated byreference). This motif has already been shown to be effective in thelocalization of reporter genes and can be used to anchor the zeta chainof the TCR. This motif is placed 5' of the variable region in order tolocalize the construct to the plasma membrane. Other modifications suchas palmitoylation can be used to anchor constructs in the plasmamembrane; for example, palmitoylation sequences from the Gprotein-coupled receptor kinase GRK6 sequence(LLQRLFSRQDCCGNCSDSEEELPTRL (SEQ ID NO:18), with the bold cysteinesbeing palmitoylated; Stoffel et al., J. Biol. Chem 269:27791 (1994));from rhodopsin (KQFRNCMLTSLCCGKNPLGD (SEQ ID NO:19); Barnstable et al.,J. Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1 protein(LNPPDESGPGCMSCKCVLS (SEQ ID NO:20); Capon et al., Nature 302:33(1983)).

In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ (SEQ ID NO:21); Dice, Ann. N.Y. Acad.Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-I(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI (SEQ ID NO:22), Uthayakumar etal., Cell. Mol. Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF (SEQ ID NO:23), Konecki et la.,Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show thetransmembrane domains in italics and the cytoplasmic targeting signalunderlined).

Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ IDNO:24); Schatz, Eur. J. Biochem. 165:1-6 (1987)); mitochondrial innermembrane sequences (yeast cytochrome c oxidase subunit IV;MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:25); Schatz, supra); mitochondrialintermembrane space sequences (yeast cytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVMAGITASTLLYADSL TAEAMTA (SEQ IDNO:26); Schatz, supra) or mitochondrial outer membrane sequences (yeast70 kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK(SEQ ID NO:27); Schatz, supra).

The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL (SEQ ID NO:28); Pelham,Royal Society London Transactions B; 1-10 (1992)) or adenovirus E3/19Kprotein (LYLSRRSFIDEKKMP (SEQ ID NO:29); Jackson et al., EMBO J. 9:3153(1990).

Furthermore, targeting sequences also include peroxisome sequences (forexample, the peroxisome matrix sequence from Luciferase; SKL; Keller etal., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21H-ras 1; LNPPDESGPGCMSCKCVLS (SEQ ID NO:30), with the bold cysteinefarnesylated; Capon, supra); geranylgeranylation sequences (for example,protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:31), with the bold cysteinesgeranylgeranylated; Farnsworth, PNAS USA 91:11963 (1994)); ordestruction sequences (cyclin B1; RTALGDIGN (SEQ ID NO:32); Klotzbucheret al., EMBO J. 1:3053 (1996)).

In a preferred embodiment, the targeting sequence is a secretory signalsequence capable of effecting the secretion of the candidate translationproduct. There are a large number of known secretory signal sequenceswhich are placed 5' to the variable peptide region, and are cleaved fromthe peptide region to effect secretion into the extracellular space.Secretory signal sequences and their transferability to unrelatedproteins are well known, e.g., Silhavy, et al. (1985) Microbiol. Rev.49, 398-418. This is particularly useful to generate a peptide capableof binding to the surface of, or affecting the physiology of, a targetcell that is other than the host cell, e.g., the cell infected with theretrovirus. In a preferred approach, a fusion product is configured tocontain, in series, secretion signal peptide-presentationstructure-randomized expression product region-presentation structure,see FIG. 3. In this manner, target cells grown in the vicinity of cellscaused to express the library of peptides, are bathed in secretedpeptide. Target cells exhibiting a physiological change in response tothe presence of a peptide, e.g., by the peptide binding to a surfacereceptor or by being internalized and binding to intracellular targets,and the secreting cells are localized by any of a variety of selectionschemes and the peptide causing the effect determined. Exemplary effectsinclude variously that of a designer cytokine (i.e., a stem cell factorcapable of causing hematopoietic stem cells to divide and maintain theirtotipotential), a factor causing cancer cells to undergo spontaneousapoptosis, a factor that binds to the cell surface of target cells andlabels them specifically, etc.

Suitable secretory sequences are known, including signals from IL-2(MYRMQLLSCIALSLALVTNS (SEQ ID NO:33); Villinger et al., J. Immunol.155:3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ IDNO:34); Roskam et al., Nucleic Acids Res. 7:30 (1979)); preproinsulin(MALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO:35); Bell et al., Nature 284:26(1980)); and influenza HA protein (MKAKLLVLLYAFVAGDQI (SEQ ID NO:36);Sekiwawa et al., PNAS 80:3563)), with cleavage between thenon-underlined-underlined junction. A particularly preferred secretorysignal sequence is the signal leader sequence from the secreted cytokineIL4, which comprises the first 24 amino acids of IL-4 as follows:MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO:37).

In a preferred embodiment, the fusion partner is a rescue sequence. Arescue sequence is a sequence which may be used to purify or isolateeither the candidate agent or the nucleic acid encoding it. Thus, forexample, peptide rescue sequences include purification sequences such asthe His₆ (SEQ ID NO:99) tag for use with Ni affinity columns and epitopetags for detection, immunoprecipitation or FACS (fluoroscence-activatedcell sorting). Suitable epitope tags include myc (for use with thecommercially available 9E10 antibody), the BSP biotinylation targetsequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.

Alternatively, the rescue sequence may be a unique oligonucleotidesequence which serves as a probe target site to allow the quick and easyisolation of the retroviral construct, via PCR, related techniques, orhybridization.

In a preferred embodiment, the fusion partner is a stability sequence toconfer stability to the candidate bioactive agent or the nucleic acidencoding it. Thus, for example, peptides may be stabilized by theincorporation of glycines after the initiation methionine (MG or MGG0),for protection of the peptide to ubiquitination as per Varshavsky'sN-End Rule, thus conferring long half-life in the cytoplasm. Similarly,two prolines at the C-terminus impart peptides that are largelyresistant to carboxypeptidase action. The presence of two glycines priorto the prolines impart both flexibility and prevent structure initiatingevents in the di-proline to be propagated into the candidate peptidestructure. Thus, preferred stability sequences are as follows: MG(X)_(n)GGPP (SEQ ID NO:38), where X is any amino acid and n is an integer of atleast four.

In one embodiment, the fusion partner is a dimerization sequence. Adimerization sequence allows the non-covalent association of one randompeptide to another random peptide, with sufficient affinity to remainassociated under normal physiological conditions. This effectivelyallows small libraries of random peptides (for example, 10⁴) to becomelarge libraries if two peptides per cell are generated which thendimerize, to form an effective library of 10⁸ (10⁴ ×10⁴). It also allowsthe formation of longer random peptides, if needed, or more structurallycomplex random peptide molecules. The dimers may be homo- orheterodimers.

Dimerization sequences may be a single sequence that self-aggregates, ortwo sequences, each of which is generated in a different retroviralconstruct. That is, nucleic acids encoding both a first random peptidewith dimerization sequence 1, and a second random peptide withdimerization sequence 2, such that upon introduction into a cell andexpression of the nucleic acid, dimerization sequence 1 associates withdimerization sequence 2 to form a new random peptide structure.

Suitable dimerization sequences will encompass a wide variety ofsequences. Any number of protein-protein interaction sites are known. Inaddition, dimerization sequences may also be elucidated using standardmethods such as the yeast two hybrid system, traditional biochemicalaffinity binding studies, or even using the present methods.

The fusion partners may be placed anywhere (i.e. N-terminal, C-terminal,internal) in the structure as the biology and activity permits.

In a preferred embodiment, the fusion partner includes a linker ortethering sequence. Linker sequences between various targeting sequences(for example, membrane targeting sequences) and the other components ofthe constructs (such as the randomized candidate agents) may bedesirable to allow the candidate agents to interact with potentialtargets unhindered. For example, when the candidate bioactive agent is apeptide, useful linkers include glycine-serine polymers (including, forexample, (GS)_(n) (SEQ ID NO:102), (GSGGS)_(n) (SEQ ID NO:39) and(GGGS)_(n) (SEQ ID NO:40), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers such as the tether for the shaker potassium channel, and a largevariety of other flexible linkers, as will be appreciated by those inthe art. Glycine-serine polymers are preferred since both of these aminoacids are relatively unstructured, and therefore may be able to serve asa neutral tether between components. Secondly, serine is hydrophilic andtherefore able to solubilize what could be a globular glycine chain.Third, similar chains have been shown to be effective in joiningsubunits of recombinant proteins such as single chain antibodies.

In addition, the fusion partners, including presentation structures, maybe modified, randomized, and/or matured to alter the presentationorientation of the randomized expression product. For example,determinants at the base of the loop may be modified to slightly modifythe internal loop peptide tertiary structure, which maintaining therandomized amino acid sequence.

In a preferred embodiment, combinations of fusion partners are used.Thus, for example, any number of combinations of presentationstructures, targeting sequences, rescue sequences, and stabilitysequences may be used, with or without linker sequences. As is morefully described below, using a base vector that contains a cloning sitefor receiving random and/or biased libraries, one can cassette invarious fusion partners 5' and 3' of the library. Table I outlines someof the possible combinations (without specifying the presentationstructures) as follows. Using V as the variable cloning site for therandom nucleic acid libraries, and representing each fusion partner byanother letter, (i.e. N for nuclear localization sequence) eachconstruct can be named as a string of representative letters reading 5'to 3' read as nucleic acid or N-terminal to C-terminal read as protein,such as NV or if cloned downstream of the variable region, VN. Asimplied here, the fusion partner sequences are cloned as cassettes intosites on either side of the variable region. C is for cytoplasmic (i.e.no localization sequence), E is a rescue sequence such as the mycepitope, G is a linker sequence (G10 is a glycine-serine chain of 10amino acids, and G20 is a glycine-serine chain of 20 amino acids), M isa myristylation sequence, N is a nuclear localization sequence, ssTM isthe signal sequence for a transmembrane anchoring sequence, TM is thetransmembrane anchoring sequence, GPI is a GPI membrane anchor sequence;S is a secretory signal sequence, etc. As will be appreciated by thosein the art, any number of combinations can be made, in addition to thoselisted below.

                  TABLE 1                                                         ______________________________________                                        cytoplasmic          C V                                                                             C E V                                                     C V E                                                                        secreted S V                                                                   S E V                                                                         S V E                                                                        myristylated M V                                                               M E V                                                                         M E G20 V                                                                    transmembrane (intracellular) ssTM V                                           ssTM V TM                                                                     ssTM V E TM                                                                   ssTM V G20 E TM                                                               ssTM V E                                                                     transmembrane (GPI linked) ssTM V G E TM                                      nuclear localization N E V                                                     N V E                                                                      ______________________________________                                    

As will be appreciated by those in the art, these modules of sequencescan be used in a large number of combinations and variations. Inaddition, as discussed herein, it is possible to have more than onevariable region in a construct, either to together form a new surface orto bring two other molecules together.

In a preferred embodiment, a candidate bioactive agent linked to apresentation structure is added at the variable region cloning site, V,above. Alternatively, no presentation structure is used, giving a "free"or "non-constrained" peptide or expression product.

Preferred embodiments include the following:

a) intracellular, membrane-anchored, linked (i.e. tethered) freepeptide: MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR-GSGGSGSGGSGSGGSGSGGSGSGGSGGG-(X)_(n)-GGPP (SEQ ID NO:41), with the secretion signal from murine CD8 in bold,the transmembrane region of CD8 in underline, and the linker, to provideflexibility (glycine) and solubility (serine) in italics. (X)_(n)represents the random peptide, where n is an integer greater than aboutsix. A preferred embodiment utilizing this structure utilizes biasedpeptides, as described below, for example using biased SH-3domain-binding peptide libraries in the non-constrained peptidestructures, since a number of surface receptor signaling systems employSH-3 domains as part of the signaling apparatus.

b) intracellular, membrane-anchored, linked coiled coil:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGSGGSGSGGSGSGGSGSGGSGSGGSGGGCAALESEVSALESEVASLESEVAAL-(X)_(n) -LAAVKSKLSAVKSKLASVKSKLAACGPP (SEQ IDNO:42), with the coiled-coil structure shown in underlined italics.

c) surface-tethered extracellular, non-constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGG-(X)_(n)-GGSGGSGSGGSGSGGSGSGGSGSGGSGGGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGGPP (SEQ ID NO:43).

d) surface-tethered, extracellular constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGGCAALESEVSALESEVASLES EVAAL-(X)_(n)-LAAVKSKLSAVKSKLASVKSKLAACGGSGGSGSGGSGSGGSGSGGSGSGGSGGGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGGPP (SEQ ID NO:44).

e) secreted, non-constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGG-(X)_(n) -GGPP (SEQ ID NO:45).

f) secreted, constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGGAALESEVSALESEVASLESE VAAL-(X)_(n)-LAAVKSKLSAVKSKLASVKSKLAACGPP (SEQ ID NO:46).

The candidate bioactive agents as described above are encoded bycandidate nucleic acids. By "candidate nucleic acids" herein is meant anucleic acid, generally RNA when retroviral delivery vehicles are used,which can be expressed to form candidate bioactive agents; that is, thecandidate nucleic acids encode the candidate bioactive agents and thefusion partners, if present. In addition, the candidate nucleic acidswill also generally contain enough extra sequence to effect translationor transcription, as necessary. For a peptide library, the candidatenucleic acid generally contains cloning sites which are placed to allowin frame expression of the randomized peptides, and any fusion partners,if present, such as presentation structures. For example, whenpresentation structures are used, the presentation structure willgenerally contain the initiating ATG, as a part of the parent vector.For a RNA library, the candidate nucleic acids are generally constructedwith an internal CMV promoter, tRNA promoter or cell specific promoterdesigned for immediate and appropriate expression of the RNA structureat the initiation site of RNA synthesis. The RNA is expressed anti-senseto the direction of retroviral synthesis and is terminated as known, forexample with an orientation specific terminator sequence. Interferencefrom upstream transcription is alleviated in the target cell with theself-inactivation deletion, a common feature of certain retroviralexpression systems.

Generally, the candidate nucleic acids are expressed within the cells toproduce expression products of the candidate nucleic acids. As outlinedabove, the expression products include translation products, i.e.peptides, or transcription products, i.e. nucleic acid.

The candidate bioactive agents and candidate nucleic acids arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. By "randomized" or grammatical equivalents herein is meantthat each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. As is more fully describedbelow, the candidate nucleic acids which give rise to the candidateexpression products are chemically synthesized, and thus may incorporateany nucleotide at any position. Thus, when the candidate nucleic acidsare expressed to form peptides, any amino acid residue may beincorporated at any position. The synthetic process can be designed togenerate randomized nucleic acids, to allow the formation of all or mostof the possible combinations over the length of the nucleic acid, thusforming a library of randomized candidate nucleic acids.

The library should provide a sufficiently structurally diversepopulation of randomized expression products to effect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Accordingly, an interactionlibrary must be large enough so that at least one of its members willhave a structure that gives it affinity for some molecule, protein, orother factor whose activity is necessary for completion of the signalingpathway. Although it is difficult to gauge the required absolute size ofan interaction library, nature provides a hint with the immune response:a diversity of 10⁷ -10⁸ different antibodies provides at least onecombination with sufficient affinity to interact with most potentialantigens faced by an organism. Published in vitro selection techniqueshave also shown that a library size of 10⁷ to 10⁸ is sufficient to findstructures with affinity for the target. A library of all combinationsof a peptide 7 to 20 amino acids in length, such as proposed here forexpression in retroviruses, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10⁷ to 10⁸ per ml of retroviral particlesthe present methods allow a "working" subset of a theoretically completeinteraction library for 7 amino acids, and a subset of shapes for the20²⁰ library. Thus, in a preferred embodiment, at least 10⁶, preferablyat least 10⁷, more preferably at least 10⁸ and most preferably at least10⁹ different expression products are simultaneously analyzed in thesubject methods. Preferred methods maximize library size and diversity.

It is important to understand that in any library system encoded byoligonucleotide synthesis one cannot have complete control over thecodons that will eventually be incorporated into the peptide structure.This is especially true in the case of codons encoding stop signals(TAA, TGA, TAG). In a synthesis with NNN as the random region, there isa 3/64, or 4.69%, chance that the codon will be a stop codon. Thus, in apeptide of 10 residues, there is an unacceptable high likelihood that46.7% of the peptides will prematurely terminate. For free peptidestructures this is perhaps not a problem. But for larger structures,such as those envisioned here, such termination will lead to sterilepeptide expression. To alleviate this, random residues are encoded asNNK, where K=T or G. This allows for encoding of all potential aminoacids (changing their relative representation slightly), but importantlypreventing the encoding of two stop residues TAA and TGA. Thus,libraries encoding a 10 amino acid peptide will have a 15.6% chance toterminate prematurely. For candidate nucleic acids which are notdesigned to result in peptide expression products, this is notnecessary.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bias is towards peptides or nucleic acidsthat interact with known classes of molecules. For example, when thecandidate bioactive agent is a peptide, it is known that much ofintracellular signaling is carried out via short regions of polypeptidesinteracting with other polypeptides through small peptide domains. Forinstance, a short region from the HIV-1 envelope cytoplasmic domain hasbeen previously shown to block the action of cellular calmodulin.Regions of the Fas cytoplasmic domain, which shows homology to themastoparan toxin from Wasps, can be limited to a short peptide regionwith death-inducing apoptotic or G protein inducing functions. Magainin,a natural peptide derived from Xenopus, can have potent anti-tumour andanti-microbial activity. Short peptide fragments of a protein kinase Cisozyme (βPKC), have been shown to block nuclear translocation of βPKCin Xenopus oocytes following stimulation. And, short SH-3 targetpeptides have been used as psuedosubstrates for specific binding to SH-3proteins. This is of course a short list of available peptides withbiological activity, as the literature is dense in this area. Thus,there is much precedent for the potential of small peptides to haveactivity on intracellular signaling cascades. In addition, agonists andantagonists of any number of molecules may be used as the basis ofbiased randomization of candidate bioactive agents as well.

Thus, a number of molecules or protein domains are suitable as startingpoints for the generation of biased randomized candidate bioactiveagents. A large number of small molecule domains are known, that confera common function, structure or affinity. In addition, as is appreciatedin the art, areas of weak amino acid homology may have strong structuralhomology. A number of these molecules, domains, and/or correspondingconsensus sequences, are known, including, but are not limited to, SH-2domains, SH-3 domains, Pleckstrin, death domains, proteasecleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf,etc. Similarly, there are a number of known nucleic acid bindingproteins containing domains suitable for use in the invention. Forexample, leucine zipper consensus sequences are known.

Where the ultimate expression product is a nucleic acid, at least 10,preferably at least 12, more preferably at least 15, most preferably atleast 21 nucleotide positions need to be randomized, with morepreferable if the randomization is less than perfect. Similarly, atleast 5, preferably at least 6, more preferably at least 7 amino acidpositions need to be randomized; again, more are preferable if therandomization is less than perfect.

In a preferred embodiment, biased SH-3 domain-bindingoligonucleotides/peptides are made. SH-3 domains have been shown torecognize short target motifs (SH-3 domain-binding peptides), about tento twelve residues in a linear sequence, that can be encoded as shortpeptides with high affinity for the target SH-3 domain. Consensussequences for SH-3 domain binding proteins have been proposed. Thus, ina preferred embodiment, oligos/peptides are made with the followingbiases 1. XXXPPXPXX (SEQ ID NO:47), wherein X is a randomized residue.2. (within the positions of residue positions 11 to -2):

11 10 9 8 7 6 5 4 3 2 1

Met Glyaa11aa10 aa9 aa8 aa7 Arg Pro Leu Pro Pro hyd 0 -1 -2 Pro hyd hydGly Gly Pro Pro STOP (SEQ ID NO:49) atg ggc nnk nnk nnk nnk nnk aga cctctg cct cca sbk ggg sbk sbk gga ggc cca cct TAA1. (SEQ ID NO:48)

In this embodiment, the N-terminus flanking region is suggested to havethe greatest effects on binding affinity and is therefore entirelyrandomized. "Hyd" indicates a bias toward a hydrophobic residue, i.e.-Val, Ala, Gly, Leu, Pro, Arg. To encode a hydrophobically biasedresidue, "sbk" codon biased structure is used. Examination of the codonswithin the genetic code will ensure this encodes generally hydrophobicresidues. s=g,c; b=t, g, c; v=a, g, c; m=a, c; k=t, g; n=a, t, g, c.

The candidate nucleic acids are introduced into the cells to screen fortransdominant bioactive agents capable of altering the phenotype of acell. By "introduced into" or grammatical equivalents herein is meantthat the nucleic acids enter the cells in a manner suitable forsubsequent expression of the nucleic acid. The method of introduction islargely dictated by the targeted cell type, discussed below. Exemplarymethods include CaPO₄ precipitation, liposome fusion, lipofectin®,electroporation, viral infection, etc. The candidate nucleic acids maystably integrate into the genome of the host cell (for example, withretroviral introduction, outlined below), or may exist eithertransiently or stably in the cytoplasm (i.e. through the use oftraditional plasmids, utilizing standard regulatory sequences, selectionmarkers, etc.). As many pharmaceutically important screens require humanor model mammalian cell targets, retroviral vectors capable oftransfecting such targets are preferred.

In a preferred embodiment, the candidate nucleic acids are part of aretroviral particle which infects the cells. Generally, infection of thecells is straightforward with the application of the infection-enhancingreagent polybrene, which is a polycation that facilitates viral bindingto the target cell. Infection can be optimized such that each cellgenerally expresses a single construct, using the ratio of virusparticles to number of cells. Infection follows a Poisson distribution.

In a preferred embodiment, the candidate nucleic acids are introducedinto the cells using retroviral vectors. Currently, the most efficientgene transfer methodologies harness the capacity of engineered viruses,such as retroviruses, to bypass natural cellular barriers to exogenousnucleic acid uptake. The use of recombinant retroviruses was pioneeredby Richard Mulligan and David Baltimore with the Psi-2 lines andanalogous retrovirus packaging systems, based on NIH 3T3 cells (see Mannet al., Cell 33:153-159 (1993), hereby incorporated by reference). Suchhelper-defective packaging lines are capable of producing all thenecessary trans proteins -gag, pol, and env- that are required forpackaging, processing, reverse transcription, and integration ofrecombinant genomes. Those RNA molecules that have in cis the ψpackaging signal are packaged into maturing virions. Retroviruses arepreferred for a number of reasons. First, their derivation is easy.Second, unlike Adenovirus-mediated gene delivery, expression fromretroviruses is long-term (adenoviruses do not integrate).Adeno-associated viruses have limited space for genes and regulatoryunits and there is some controversy as to their ability to integrate.Retroviruses therefore offer the best current compromise in terms oflong-term expression, genomic flexibility, and stable integration, amongother features. The main advantage of retroviruses is that theirintegration into the host genome allows for their stable transmissionthrough cell division. This ensures that in cell types which undergomultiple independent maturation steps, such as hematopoietic cellprogression, the retrovirus construct will remain resident and continueto express.

A particularly well suited retroviral transfection system is describedin Mann et al., supra: Pear et al., PNAS USA 90(18):8392-6 (1993);Kitamura et al., PNAS USA 92:9146-9150 (1995); Kinsella et al., HumanGene Therapy 7:1405-1413; Hofmann et al., PNAS USA 93:5185-5190; Choateet al., Human Gene Therapy 7:2247 (1996); and WO 94/19478; andreferences cited therein, all of which are incorporated by reference.

In one embodiment of the invention, the library is generated in aretrovirus DNA construct backbone, as is generally described in theexamples. Standard oligonucleotide synthesis is done to generate therandom portion of the candidate bioactive agent, using techniques wellknown in the art (see Eckstein, Oligonucleotides and Analogues, APractical Approach, IRL Press at Oxford University Press, 1991);libraries may be commercially purchased. Libraries with up to 10⁹ uniquesequences can be readily genera t ed in such DNA backbones. Aftergeneration of the DNA library, the library is cloned into a firstprimer. The first primer serves as a "cassette", which is inserted intothe retroviral construct. The first primer generally contains a numberof elements, including for example, the require d regulatory sequences(e.g. translation, transcription, promoters, etc), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc.

A second primer is then added, which generally consists of some or allof the complementarity region to prime the first primer and optionalnecessary sequences for a second unique restriction site for subcloning.DNA polymerase is added to make double-stranded oligonucleotides. Thedouble-stranded oligonucletides are cleaved with the appropriatesubcloning restriction endonucleases and subcloned into the targetretroviral vectors, described below.

Any number of suitable retroviral vectors may be used. Generally, theretroviral vectors may include: selectable marker genes under thecontrol of internal ribosome entry sites (IRES), which allows forbioistronic operons and thus greatly facilitates the selection of cellsexpressing peptides at uniformly high levels; and promoters drivingexpression of a second gene, placed in sense or anti-sense relative tothe 5' LTR. Suitable selection genes include, but are not limited to,neomycin, blastocidin, bleomycin, puromycin, and hygromycin resistancegenes, as well as self-fluorescent markers such as green fluoroscentprotein, enzymatic markers such as lacZ, and surface proteins such asCD8, etc.

Preferred vectors include a vector based on the murine stem cell virus(MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and a modified MFGvirus (Rivere et al., Genetics 92:6733 (1995)), and pBABE, outlined inthe examples. A general schematic of the retroviral construct isdepicted in FIG. 4.

The retroviruses may include inducible and constitutive promoters. Forexample, there are situations wherein it is necessary to induce peptideexpression only during certain phases of the selection process. Forinstance, a scheme to provide pro-inflammatory cytokines in certaininstances must include induced expression of the peptides. This isbecause there is some expectation that over-expressed pro-inflammatorydrugs might in the long-term be detrimental to cell growth. Accordingly,constitutive expression is undesirable, and the peptide is only turnedon during that phase of the selection process when the phenotype isrequired, and then shut the peptide down by turning off the retroviralexpression to confirm the effect or ensure long-term survival of theproducer cells. A large number of both inducible and constitutivepromoters are known.

In addition, it is possible to configure a retroviral vector to allowinducible expression of retroviral inserts after integration of a singlevector in target cells; importantly, the entire system is containedwithin the single retrovirus. Tet-inducible retroviruses have beendesigned incorporating the Self-inactivating (SIN) feature of 3' LTRenhancer/promoter retroviral deletion mutant (Hoffman et al., PNAS USA93:5185 (1996)). Expression of this vector in cells is virtuallyundetectable in the presence of tetracycline or other active analogs.However, in the absence of Tet, expression is turned on to maximumwithin 48 hours after induction, with uniform increased expression ofthe whole population of cells that harbor the inducible retrovirus,indicating that expression is regulated uniformly within the infectedcell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

In this manner the primers create a library of fragments, eachcontaining a different random nucleotide sequence that may encode adifferent peptide. The ligation products are then transformed intobacteria, such as E. coli, and DNA is prepared from the resultinglibrary, as is generally outlined in Kitamura, PNAS USA 92:9146-9150(1995), hereby expressly incorporated by reference.

Delivery of the library DNA into a retroviral packaging system resultsin conversion to infectious virus. Suitable retroviral packaging systemcell lines include, but are not limited to, the Bing and BOSC23 celllines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., PNAS USA 93:11658 (1996), Kinsella et al., Human GeneTherapy 7:1405 (1996), all of which are incorporated by reference.

Preferred systems include PhiNX-eco and PhiNX-ampho or similar celllines, which are two cells lines as follows. The cell lines are based onthe BING and BOSC23 cell lines described in WO 94/19478, which are basedon the 293T cell line (a human embryonic kidney line transformed withadenovirus E1a and carrying a temperature sensitive T antigenco-selected with neomycin). The unique feature of this cell line is thatit is highly transfectable with either calcium phosphate mediatedtransfection or lipid-based transfection protocols--greater than 50% of293T cells can be transiently transfected with plasmid DNA. Thus, thecell line could be a cellular milieu in which retroviral structuralproteins and genomic viral RNA could brought together rapidly forcreation of helper-defective virus. 293T cells were therefore engineeredwith stably integrated defective constructs capable of producinggag-pol, and envelope protein for either ecotropic or amphotropicviruses. These lines were called BOSC23 and Bing, respectively. Theutility of these lines was that one could produce small amounts ofrecombinant virus transiently for use in small-scale experimentation.The lines offered advantages over previous stable systems in that viruscould be produced in days rather than months.

Two problems became apparent with these first generation lines over thetwo years they have been in wide use. First, gag-pol and envelopeexpression was unstable and the lines required vigilant checking forretroviral production capacity; second the structure of the vectors usedfor protein production were not considered fully "safe" for helper virusproduction; and third, one of the lines was shown to be inadvertentlycarrying a hygromycin-containing retrovirus. Although the BING andBOSC23 lines are useful in the present invention, all of thesepotentially problematic issues are addressed in the PhiNXsecond-generation lines. These lines are based on 293T cells as well,with the following improvements. First, the ability to monitor gag-polproduction on a cell-by cell basis was made by introducing an IRES-CD8surface marker expression cassette downstream of the reading frame ofthe gag-pol construct (other surface markers besides CD8 are alsouseful). IRES (internal ribosome entry site) sequences allow secondaryor tertiary protein translation from a single mRNA transcript. Thus, CD8expression is a direct reflection of intracellular gag-pol and thestability of the producer cell population's ability to produce gag-polcan be readily monitored by flow cytometry. Second, for both the gag-poland envelope constructs non-Moloney promoters were used to minimizerecombination potential with introduced retroviral constructs, anddifferent promoters for gag-pol and envelope were used to minimize theirinter-recombination potential. The promoters used were CMV and RSV. Twocell lines were created, PHEONIX-ECO and PHEONIX-AMPHO. Gag-pol wasintroduced with hygromycin as the co-selectable marker and the envelopeproteins were introduced with diphtheria resistance as the co-selectablemarker. Finally, the cells were screened to find a relatively rare celltype that produced gag-pol and env in a uniform distribution, althoughthis is not required. In addition, a line termed PHEONIX-gp has beenproduced that expresses only gag-pol. This line is available for furtherpseudotyping of retroviral virions with other envelope proteins such asgibbon ape leukemia virus envelope or Vesicular Stomatitis VSV-Gprotein, Xenotropic, or retargeting envelopes can also be added.

Both PHEONIX-ECO and PHEONIX-AMPHO were tested for helper virusproduction and established as being helper-virus free. Both lines cancarry episomes for the creation of stable cell lines which can be usedto produce retrovirus. Both lines are readily testable by flow cytometryfor stability of gag-pol (CD8) and envelope expression; after severalmonths of testing the lines appear stable, and do not demonstrate lossof titre as did the first-generation lines BOSC23 and Bing (partly dueto the choice of promoters driving expression of gag-pol and envelope).Both lines can also be used to transiently produce virus in a few days.Thus, these new lines are fully compatible with transient, episomalstable, and library generation for retroviral gene transfer experiments.Finally, the titres produced by these lines have been tested. Usingstandard polybrene-enhanced retroviral infection, titres approaching orabove 10⁷ per ml were observed for both PHEONIX-eco and PHEONIX-amphowhen carrying episomal constructs. When transiently produced virus ismade, titres are usually 1/2 to 1/3 that value.

These lines are helper-virus free, carry episomes for long-term stableproduction of retrovirus, stably produce gag-pol and env, and do notdemonstrate loss of viral titre over time. In addition, PhiNX-eco andPhiNX-ampho are capable of producing titres approaching or above 10⁷ perml when carrying episomal constructs, which, with concentration ofvirus, can be enhanced to 10⁸ to 10⁹ per ml.

In a preferred embodiment, the cell lines disclosed above, and the othermethods for producing retrovirus, are useful for production of virus bytransient transfection. The virus can either be used directly or be usedto infect another retroviral producer cell line for "expansion" of thelibrary.

Concentration of virus may be done as follows. Generally, retrovirusesare titred by applying retrovirus-containing supernatant onto indicatorcells, such as NIH3T3 cells, and then measuring the percentage of cellsexpressing phenotypic consequences of infection. The concentration ofthe virus is determined by multiplying the percentage of cells infectedby the dilution factor involved, and taking into account the number oftarget cells available to obtain a relative titre. If the retroviruscontains a reporter gene, such as lacZ, then infection, integration, andexpression of the recombinant virus is measured by histological stainingfor lacZ expression or by flow cytometry (FACS). In general, retroviraltitres generated from even the best of the producer cells do not exceed10⁷ per ml, unless concentration by relatively expensive or exoticapparatus. However, as it has been recently postulated that since aparticle as large as a retrovirus will not move very far by brownianmotion in liquid, fluid dynamics predicts that much of the virus nevercomes in contact with the cells to initiate the infection process.However, if cells are grown or placed on a porous filter and retrovirusis allowed to move past cells by gradual gravitometric flow, a highconcentration of virus around cells can be effectively maintained at alltimes. Thus, up to a ten-fold higher infectivity by infecting cells on aporous membrane and allowing retrovirus supernatant to flow past themhas been seen. This should allow titres of 10⁹ after concentration.

The candidate nucleic acids, as part of the retroviral construct, areintroduced into the cells to screen for transdominant bioactive agentscapable of altering the phenotype of a cell.

As will be appreciated by those in the art, the type of cells used inthe present invention can vary widely. Basically, any mammalian cellsmay be used, with mouse, rat, primate and human cells being particularlypreferred, although as will be appreciated by those in the art,modifications of the system by pseudotyping allows all eukaryotic cellsto be used, preferably higher eukaryotes. As is more fully describedbelow, a screen will be set up such that the cells exhibit a selectablephenotype in the presence of a bioactive agent. As is more fullydescribed below, cell types implicated in a wide variety of diseaseconditions are particularly useful, so long as a suitable screen may bedesigned to allow the selection of cells that exhibit an alteredphenotype as a consequence of the presence of a transdominant bioactiveagent within the cell.

Accordingly, suitable cell types include, but are not limited to, tumorcells of all types (particularly melanoma, myeloid leukemia, carcinomasof the lung, breast, ovaries, colon, kidney, prostate, pancreas andtestes), cardiomyocytes, endothelial cells, epithelial cells,lymphocytes (T-cell and B cell) , mast cells, eosinophils, vascularintimal cells, hepatocytes, leukocytes including mononuclear leukocytes,stem cells such as haemopoetic, neural, skin, lung, kidney, liver andmyocyte stem cells (for use in screening for differentiation andde-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

In one embodiment, the cells may be genetically engineered, that is,contain erogenous nucleic acid, for example, to contain targetmolecules.

In a preferred embodiment, a first plurality of cells is screened. Thatis, the cells into which the candidate nucleic acids are introduced arescreened for an altered phenotype. Thus, in this embodiment, the effectof the transdominant bioactive agent is seen in the same cells in whichit is made; i.e. an autocrine effect.

By a "plurality of cells" herein is meant roughly from about 10³ cellsto 10⁸ or 10⁹, with from 10⁶ to 10⁸ being preferred. This plurality ofcells comprises a cellular library, wherein generally each cell withinthe library contains a member of the retroviral molecular library, i.e.a different candidate nucleic acid, although as will be appreciated bythose in the art, some cells within the library may not contain aretrovirus, and some may contain more than one. When methods other thanretroviral infection are used to introduce the candidate nucleic acidsinto a plurality of cells, the distribution of candidate nucleic acidswithin the individual cell members of the cellular library may varywidely, as it is generally difficult to control the number of nucleicacids which enter a cell during electroporation, etc.

In a preferred embodiment, the candidate nucleic acids are introducedinto a first plurality of cells, and the effect of the candidatebioactive agents is screened in a second or third plurality of cells,different from the first plurality of cells, i.e. generally a differentcell type. That is, the effect of the transdominant bioactive agents isdue to an extracellular effect on a second cell; i.e. an endocrine orparacrine effect. This is done using standard techniques. The firstplurality of cells may be grown in or on one media, and the media isallowed to touch a second plurality of cells, and the effect measured.Alternatively, there may be direct contact between the cells. Thus,"contacting" is functional contact, and includes both direct andindirect. In this embodiment, the first plurality of cells may or maynot be screened.

If necessary, the cells are treated to conditions suitable for theexpression of the candidate nucleic acids (for example, when induciblepromoters are used), to produce the candidate expression products,either translation or transcription products.

Thus, the methods of the present invention comprise introducing amolecular library of randomized candidate nucleic acids into a pluralityof cells, a cellular library. Each of the nucleic acids comprises adifferent, generally randomized, nucleotide sequence. The plurality ofcells is then screened, as is more fully outlined below, for a cellexhibiting an altered phenotype. The altered phenotype is due to thepresence of a transdominant bioactive agent.

By "altered phenotype" or "changed physiology" or other grammaticalequivalents herein is meant that the phenotype of the cell is altered insome way, preferably in some detectable and/or measurable way. As willbe appreciated in the art, a strength of the present invention is thewide variety of cell types and potential phenotypic changes which may betested using the present methods. Accordingly, any phenotypic changewhich may be observed, detected, or measured may be the basis of thescreening methods herein. Suitable phenotypic changes include, but arenot limited to: gross physical changes such as changes in cellmorphology, cell growth, cell viability, adhesion to substrates or othercells, and cellular density; changes in the expression of one or moreRNAs, proteins, lipids, hormones, cytokines, or other molecules; changesin the equilibrium state (i.e. half-life) or one or more RNAs, proteins,lipids, hormones, cytokines, or other molecules; changes in thelocalization of one or more RNAs, proteins, lipids, hormones, cytokines,or other molecules; changes in the bioactivity or specific activity ofone or more RNAs, proteins, lipids, hormones, cytokines, receptors, orother molecules; changes in the secretion of ions, cytokines, hormones,growth factors, or other molecules; alterations in cellular membranepotentials, polarization, integrity or transport; changes ininfectivity, susceptibility, latency, adhesion, and uptake of virusesand bacterial pathogens; etc. By "capable of altering the phenotype"herein is meant that the bioactive agent can change the phenotype of thecell in some detectable and/or measurable way.

The altered phenotype may be detected in a wide variety of ways, as isdescribed more fully below, and will generally depend and correspond tothe phenotype that is being changed. Generally, the changed phenotype isdetected using, for example: microscopic analysis of cell morphology;standard cell viability assays, including both increased cell death andincreased cell viability, for example, cells that are now resistant tocell death via virus, bacteria, or bacterial or synthetic toxins;standard labeling assays such as fluorometric indicator assays for thepresence or level of a particular cell or molecule, including FACS orother dye staining techniques; biochemical detection of the expressionof target compounds after killing the cells; etc. In some cases, as ismore fully described herein, the altered phenotype is detected in thecell in which the randomized nucleic acid was introduced; in otherembodiments, the altered phenotype is detected in a second cell which isresponding to some molecular signal from the first cell.

An altered phenotype of a cell indicates the presence of a transdominantbioactive agent. By "transdominant" herein is meant that the bioactiveagent indirectly causes the altered phenotype by acting on a secondmolecule, which leads to an altered phenotype. That is, a transdominantexpression product has an effect that is not in cis, i.e., a trans eventas defined in genetic terms or biochemical terms. A transdominant effectis a distinguishable effect by a molecular entity (i.e., the encodedpeptide or RNA) upon some separate and distinguishable target; that is,not an effect upon the encoded entity itself. As such, transdominanteffects include many well-known effects by pharmacologic agents upontarget molecules or pathways in cells or physiologic systems; forinstance, the β-lactam antibiotics have a transdominant effect uponpeptidoglycan synthesis in bacterial cells by binding to penicillinbinding proteins and disrupting their functions. An exemplarytransdominant effect by a peptide is the ability to inhibit NF-KBsignaling by binding to IKB-a at a region critical for its function,such that in the presence of sufficient amounts of the peptide (ormolecular entity), the signaling pathways that normally lead to theactivation of NF-KB through phosphorylation and/or degradation of IKB-αare inhibited from acting at IKB-a because of the binding of the peptideor molecular entity. In another instance, signaling pathways that arenormally activated to secrete IgE are inhibited in the presence ofpeptide. Or, signaling pathways in adipose tissue cells, normallyquiescent, are activated to metabolize fat. Or, in the presence of apeptide, intracellular mechanisms for the replication of certainviruses, such as HIV-I, or Herpes viridae family members, or RespiratorySyncytial Virus, for example, are inhibited.

A transdominant effect upon a protein or molecular pathway is clearlydistinguishable from randomization, change, or mutation of a sequencewithin a protein or molecule of known or unknown function to enhance ordiminish a biochemical ability that protein or molecule alreadymanifests. For instance, a protein that enzymatically cleaves β-lactamantibiotics, a β-lactamase, could be enhanced or diminished in itsactivity by mutating sequences internal to its structure that enhance ordiminish the ability of this enzyme to act upon and cleave β-lactamantibiotics. This would be called a cis mutation to the protein. Theeffect of this protein upon β-lactam antibiotics is an activity theprotein already manifests, to a distinguishable degree. Similarly, amutation in the leader sequence that enhanced the export of this proteinto the extracellular spaces wherein it might encounter β-lactammolecules more readily, or a mutation within the sequence that enhancethe stability of the protein, would be termed cis mutations in theprotein. For comparison, a transdominant effector of this protein wouldinclude an agent, independent of the β-lactamase, that bound to theβ-lactamase in such a way that it enhanced or diminished the function ofthe β-lactamase by virtue of its binding to P-lactamase.

In general, cis-effects are effects within molecules wherein elementsthat are interacting are covalently joined to each other although theseelements might individually manifest themselves as separable domains.Trans-effects (transdominant in that under some cellular conditions thedesired effect is manifested) are those effects between distinctmolecular entities, such that molecular entity A, not covalently linkedto molecular entity B, binds to or otherwise has an effect upon theactivities of entity B. As such, most known pharmacological agents aretransdominant effectors.

In a preferred embodiment, once a cell with an altered phenotype isdetected, the cell is isolated from the plurality which do not havealtered phenotypes. This may be done in any number of ways, as is knownin the art, and will in some instances depend on the assay or screen.Suitable isolation techniques include, but are not limited to, FACS,lysis selection using complement, cell cloning, scanning by Fluorimager,expression of a "survival" protein, induced expression of a cell surfaceprotein or other molecule that can be rendered fluorescent or taggablefor physical isolation; expression of an enzyme that changes anon-fluorescent molecule to a fluoroscent one; overgrowth against abackground of no or slow growth; death of cells and isolation of DNA orother cell vitality indicator dyes, etc.

In a preferred embodiment, the candidate nucleic acid and/or thebioactive agent is isolated from the positive cell. This may be done ina number of ways. In a preferred embodiment, primers complementary toDNA regions common to the retroviral constructs, or to specificcomponents of the library such as a rescue sequence, defined above, areused to "rescue" the unique random sequence. Alternatively, thebioactive agent is isolated using a rescue sequence. Thus, for example,rescue sequences comprising epitope tags or purification sequences maybe used to pull out the bioactive agent, using immunoprecipitation oraffinity columns. In some instances, as is outlined below, this may alsopull out the primary target molecule, if there is a sufficiently strongbinding interaction between the bioactive agent and the target molecule.Alternatively, the peptide may be detected using mass spectroscopy.

Once rescued, the sequence of the bioactive agent and/or bioactivenucleic acid is determined. This information can then be used in anumber of ways.

In a preferred embodiment, the bioactive agent is resynthesized andreintroduced into the target cells, to verify the effect. This may bedone using retroviruses, or alternatively using fusions to the HIV-1 Tatprotein, and analogs and related proteins, which allows very high uptakeinto target cells. See for example, Fawell et al., PNAS USA 91:664(1994); Frankel et al., Cell 55:1189 (1988); Savion et al., J. Biol.Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444 (1994);and Baldin et al., EMBO J. 9:1511 (1990), all of which are incorporatedby reference.

In a preferred embodiment, the sequence of a bioactive agent is used togenerate more candidate bioactive agents. For example, the sequence ofthe bioactive agent may be the basis of a second round of (biased)randomization, to develop bioactive agents with increased or alteredactivities. Alternatively, the second round of randomization may changethe affinity of the bioactive agent. Furthermore, it may be desirable toput the identified random region of the bioactive agent into otherpresentation structures, or to alter the sequence of the constant regionof the presentation structure, to alter the conformation/shape of thebioactive agent. It may also be desirable to "walk" around a potentialbinding site, in a manner similar to the mutagenesis of a bindingpocket, by keeping one end of the ligand region constant and randomizingthe other end to shift the binding of the peptide around.

In a preferred embodiment, either the bioactive agent or the bioactivenucleic acid encoding it is used to identify target molecules, i.e. themolecules with which the bioactive agent interacts. As will beappreciated by those in the art, there may be primary target molecules,to which the bioactive agent binds or acts upon directly, and there maybe secondary target molecules, which are part of the signalling pathwayaffected by the bioactive agent; these might be termed "validatedtargets".

In a preferred embodiment, the bioactive agent is used to pull outtarget molecules. For example, as outlined herein, if the targetmolecules are proteins, the use of epitope tags or purificationsequences can allow the purification of primary target molecules viabiochemical means (co-immunoprecipitation, affinity columns, etc.).Alternatively, the peptide, when expressed in bacteria and purified, canbe used as a probe against a bacterial cDNA expression library made frommRNA of the target cell type. Or, peptides can be used as "bait" ineither yeast or mammalian two or three hybrid systems. Such interactioncloning approaches have been very useful to isolate DNA-binding proteinsand other interacting protein components. The peptide(s) can be combinedwith other pharmacologic activators to study the epistatic relationshipsof signal transduction pathways in question. It is also possible tosynthetically prepare labeled peptide bioactive agent and use it toscreen a cDNA library expressed in bacteriophage for those cDNAs whichbind the peptide. Furthermore, it is also possible that one could usecDNA cloning via retroviral libraries to "complement" the effect inducedby the peptide. In such a strategy, the peptide would be required to bestochiometrically titrating away some important factor for a specificsignaling pathway. If this molecule or activity is replenished byover-expression of a cDNA from within a cDNA library, then one can clonethe target. Similarly, cDNAs cloned by any of the above yeast orbacteriophage systems can be reintroduced to mammalian cells in thismanner to confirm that they act to complement function in the system thepeptide acts upon.

Once primary target molecules have been identified, secondary targetmolecules may be identified in the same manner, using the primary targetas the "bait". In this manner, signalling pathways may be elucidated.Similarly, bioactive agents specific for secondary target molecules mayalso be discovered, to allow a number of bioactive agents to act on asingle pathway, for example for combination therapies.

The screening methods of the present invention may be useful to screen alarge number of cell types under a wide variety of conditions.Generally, the host cells are cells that are involved in disease states,and they are tested or screened under conditions that normally result inundesirable consequences on the cells. When a suitable bioactive agentis found, the undesirable effect may be reduced or eliminated.Alternatively, normally desirable consequences may be reduced oreliminated, with an eye towards elucidating the cellular mechanismsassociated with the disease state or signalling pathway.

In a preferred embodiment, the present methods are useful in cancerapplications. The ability to rapidly and specifically kill tumor cellsis a cornerstone of cancer chemotherapy. In general, using the methodsof the present invention, random libraries can be introduced into anytumor cell (primary or cultured), and peptides identified which bythemselves induce apoptosis, cell death, loss of cell division ordecreased cell growth. This may be done de novo, or by biasedrandomization toward known peptide agents, such as angiostatin, whichinhibits blood vessel wall growth. Alternatively, the methods of thepresent invention can be combined with other cancer therapeutics (e.g.drugs or radiation) to sensitize the cells and thus induce rapid andspecific apoptosis, cell death, loss of cell division or decreased cellgrowth after exposure to a secondary agent. Similarly, the presentmethods may be used in conjunction with known cancer therapeutics toscreen for agonists to make the therapeutic more effective or lesstoxic. This is particularly preferred when the chemotherapeutic is veryexpensive to produce such as taxol.

Known oncogenes such as v-AbI, v-Src, v-Ras, and others, induce atransformed phenotype leading to abnormal cell growth when transfectedinto certain cells. This is also a major problem with micro-metastases.Thus, in a preferred embodiment, non-transformed cells can betransfected with these oncogenes, and then random libraries introducedinto these cells, to select for bioactive agents which reverse orcorrect the transformed state. One of the signal features of oncogenetransformation of cells is the loss of contact inhibition and theability to grow in soft-agar. When transforming viruses are constructedcontaining v-Abl, v-Src, or v-Ras in IRES-puro retroviral vectors,infected into target 3T3 cells, and subjected to puromycin selection,all of the 3T3 cells hyper-transform and detach from the plate. Thecells may be removed by washing with fresh medium. This can serve as thebasis of a screen, since cells which express a bioactive agent willremain attached to the plate and form colonies.

Similarly, the growth and/or spread of certain tumor types is enhancedby stimulatory responses from growth factors and cytokines (PDGF, EGF,Heregulin, and others) which bind to receptors on the surfaces ofspecific tumors. In a preferred embodiment, the methods of the inventionare used to inhibit or stop tumor growth and/or spread, by findingbioactive agents capable of blocking the ability of the growth factor orcytokine to stimulate the tumor cell. The introduction of randomlibraries into specific tumor cells with the addition of the growthfactor or cytokine, followed by selection of bioactive agents whichblock the binding, signaling, phenotypic and/or functional responses ofthese tumor cells to the growth factor or cytokine in question.

Similarly, the spread of cancer cells (invasion and metastasis) is asignificant problem limiting the success of cancer therapies. Theability to inhibit the invasion and/or migration of specific tumor cellswould be a significant advance in the therapy of cancer. Tumor cellsknown to have a high metastatic potential (for example, melanoma, lungcell carcinoma, breast and ovarian carcinoma) can have random librariesintroduced into them, and peptides selected which in a migration orinvasion assay, inhibit the migration and/or invasion of specific tumorcells. Particular applications for inhibition of the metastaticphenotype, which could allow a more specific inhibition of metastasis,include the metastasis suppressor gene NM23, which codes for adinucleoside diphosphate kinase. Thus intracellular peptide activatorsof this gene could block metastasis, and a screen for its upregulation(by fusing it to a reporter gene) would be of interest. Many oncogenesalso enhance metastasis. Peptides which inactivate or counteract mutatedRAS oncogenes, v-MOS, v-RAF, A-RAF, v-SRC, v-FES, and v-FMS would alsoact as anti-metastatics. Peptides which act intracellularly to block therelease of combinations of proteases required for invasion, such as thematrix metalloproteases and urokinase, could also be effectiveantimetastatics.

In a preferred embodiment, the random libraries of the present inventionare introduced into tumor cells known to have inactivated tumorsuppressor genes, and successful reversal by either reactivation orcompensation of the knockout would be screened by restoration of thenormal phenotype. A major example is the reversal of p53-inactivatingmutations, which are present in 50% or more of all cancers. Since p53'sactions are complex and involve its action as a transcription factor,there are probably numerous potential ways a peptide or small moleculederived from a peptide could reverse the mutation. One example would beupregulation of the immediately downstream cyclin-dependent kinasep21CIP1/WAF1. To be useful such reversal would have to work for many ofthe different known p53 mutations. This is currently being approached bygene therapy; one or more small molecules which do this might bepreferable.

Another example involves screening of bioactive agents which restore theconstitutive function of the brca-1 or brca-2 genes, and other tumorsuppressor genes important in breast cancer such as the adenomatouspolyposis coli gene (APC) and the Drosophila discs-large gene (Dlg),which are components of cell--cell junctions. Mutations of brca-1 areimportant in hereditary ovarian and breast cancers, and constitute anadditional application of the present invention.

In a preferred embodiment, the methods of the present invention are usedto create novel cell lines from cancers from patients. A retrovirallydelivered short peptide which inhibits the final common pathway ofprogrammed cell death should allow for short- and possibly long-termcell lines to be established. Conditions of in vitro culture andinfection of human leukemia cells will be established. There is a realneed for methods which allow the maintenance of certain tumor cells inculture long enough to allow for physiological and pharmacologicalstudies. Currently, some human cell lines have been established by theuse of transforming agents such as Epstein-Barr virus that considerablyalters the existing physiology of the cell. On occasion, cells will growon their own in culture but this is a random event. Programmed celldeath (apoptosis) occurs via complex signaling pathways within cellsthat ultimately activate a final common pathway producing characteristicchanges in the cell leading to a non-inflammatory destruction of thecell. It is well known that tumor cells have a high apoptotic index, orpropensity to enter apoptosis in vivo. When cells are placed in culture,the in vivo stimuli for malignant cell growth are removed and cellsreadily undergo apoptosis. The objective would be to develop thetechnology to establish cell lines from any number of primary tumorcells, for example primary human leukemia cells, in a reproduciblemanner without altering the native configuration of the signalingpathways in these cells. By introducing nucleic acids encoding peptideswhich inhibit apoptosis, increased cell survival in vitro, and hence theopportunity to study signalling transduction pathways in primary humantumor cells, is accomplished. In addition, these methods may be used forculturing primary cells, i.e. non-tumor cells.

In a preferred embodiment, the present methods are useful incardiovascular applications. In a preferred embodiment, cardiomyocytesmay be screened for the prevention of cell damage or death in thepresence of normally injurious conditions, including, but not limitedto, the presence of toxic drugs (particularly chemotherapeutic drugs),for example, to prevent heart failure following treatment withadriamycin; anoxia, for example in the setting of coronary arteryocclusion; and autoimmune cellular damage by attack from activatedlymphoid cells (for example as seen in post viral myocarditis andlupus). Candidate bioactive agents are inserted into cardiomyocytes, thecells are subjected to the insult, and bioactive agents are selectedthat prevent any or all of: apoptosis; membrane depolarization (i.e.decrease arrythmogenic potential of insult); cell swelling; or leakageof specific intracellular ions, second messengers and activatingmolecules (for example, arachidonic acid and/or lysophosphatidic acid).

In a preferred embodiment, the present methods are used to screen fordiminished arrhythmia potential in cardiomyocytes. The screens comprisethe introduction of the candidate nucleic acids encoding candidatebioactive agents, followed by the application of arrythmogenic insults,with screening for bioactive agents that block specific depolarizationof cell membrane. This may be detected using patch clamps, or viafluorescence techniques). Similarly, channel activity (for example,potassium and chloride channels) in cardiomyocytes could be regulatedusing the present methods in order to enhance contractility and preventor diminish arrhythmias.

In a preferred embodiment, the present methods are used to screen forenhanced contractile properties of cardiomyocytes and diminish heartfailure potential. The introduction of the libraries of the inventionfollowed by measuring the rate of change of myosinpolymerization/depolymerization using fluorescent techniques can bedone. Bioactive agents which increase the rate of change of thisphenomenon can result in a greater contractile response of the entiremyocardium, similar to the effect seen with digitalis.

In a preferred embodiment, the present methods are useful to identifyagents that will regulate the intracellular and sarcolemmal calciumcycling in cardiomyocytes in order to prevent arrhythmias. Bioactiveagents are selected that regulate sodium-calcium exchange, sodium protonpump function, and regulation of calcium-ATPase activity.

In a preferred embodiment, the present methods are useful to identifyagents that diminish embolic phenomena in arteries and arteriolesleading to strokes (and other occlusive events leading to kidney failureand limb ischemia) and angina precipitating a myocardial infarct areselected. For example, bioactive agents which will diminish the adhesionof platelets and leukocytes, and thus diminish the occlusion events.Adhesion in this setting can be inhibited by the libraries of theinvention being inserted into endothelial cells (quiescent cells, oractivated by cytokines, i.e. IL-1, and growth factors, i.e. PDGF / EGF)and then screening for peptides that either: 1) downregulate adhesionmolecule expression on the surface of the endothelial cells (bindingassay); 2) block adhesion molecule activation on the surface of thesecells (signaling assay); or 3) release in an autocrine manner peptidesthat block receptor binding to the cognate receptor on the adheringcell.

Embolic phenomena can also be addressed by activating proteolyticenzymes on the cell surfaces of endothelial cells, and thus releasingactive enzyme which can digest blood clots. Thus, delivery of thelibraries of the invention to endothelial cells is done, followed bystandard fluorogenic assays, which will allow monitoring of proteolyticactivity on the cell surface towards a known substrate. Bioactive agentscan then be selected which activate specific enzymes towards specificsubstrates.

In a preferred embodiment, arterial inflammation in the setting ofvasculitis and post-infarction can be regulated by decreasing thechemotactic responses of leukocytes and mononuclear leukocytes. This canbe accomplished by blocking chemotactic receptors and their respondingpathways on these cells. Candidate bioactive libraries can be insertedinto these cells, and the chemotactic response to diverse chemokines(for example, to the IL-8 family of chemokines, RANTES) inhibited incell migration assays.

In a preferred embodiment, arterial restenosis following coronaryangioplasty can be controlled by regulating the proliferation ofvascular intimal cells and capillary and/or arterial endothelial cells.Candidate bioactive agent libraries can be inserted into these celltypes and their proliferation in response to specific stimuli monitored.One application may be intracellular peptides which block the expressionor function of c-myc and other oncogenes in smooth muscle cells to stoptheir proliferation. A second application may involve the expression oflibraries in vascular smooth muscle cells to selectively induce theirapoptosis. Application of small molecules derived from these peptidesmay require targeted drug delivery; this is available with stents,hydrogel coatings, and infusion-based catheter systems. Peptides whichdownregulate endothelin-1A receptors or which block the release of thepotent vasoconstrictor and vascular smooth muscle cell mitogenendothelin-1 may also be candidates for therapeutics. Peptides can beisolated from these libraries which inhibit growth of these cells, orwhich prevent the adhesion of other cells in the circulation known torelease autocrine growth factors, such as platelets (PDGF) andmononuclear leukocytes.

The control of capillary and blood vessel growth is an important goal inorder to promote increased blood flow to ischemic areas (growth), or tocut-off the blood supply (angiogenesis inhibition) of tumors. Candidatebioactive agent libraries can be inserted into capillary endothelialcells and their growth monitored. Stimuli such as low oxygen tension andvarying degrees of angiogenic factors can regulate the responses, andpeptides isolated that produce the appropriate phenotype. Screening forantagonism of vascular endothelial cell growth factor, important inangiogenesis, would also be useful.

In a preferred embodiment, the present methods are useful in screeningfor decreases in atherosclerosis producing mechanisms to find peptidesthat regulate LDL and HDL metabolism. Candidate libraries can beinserted into the appropriate cells (including hepatocytes, mononuclearleukocytes, endothelial cells) and peptides selected which lead to adecreased release of LDL or diminished synthesis of LDL, or converselyto an increased release of HDL or enhanced synthesis of HDL. Bioactiveagents can also be isolated from candidate libraries which decrease theproduction of oxidized LDL, which has been implicated in atherosclerosisand isolated from atherosclerotic lesions. This could occur bydecreasing its expression, activating reducing systems or enzymes, orblocking the activity or production of enzymes implicated in productionof oxidized LDL, such as 15-lipoxygenase in macrophages.

In a preferred embodiment, the present methods are used in screens toregulate obesity via the control of food intake mechanisms ordiminishing the responses of receptor signaling pathways that regulatemetabolism. Bioactive agents that regulate or inhibit the responses ofneuropeptide Y (NPY), cholecystokinin and galanin receptors, areparticularly desirable. Candidate libraries can be inserted into cellsthat have these receptors cloned into them, and inhibitory peptidesselected that are secreted in an autocrine manner that block thesignaling responses to galanin and NPY. In a similar manner, peptidescan be found that regulate the leptin receptor.

In a preferred embodiment, the present methods are useful inneurobiology applications. Candidate libraries may be used for screeningfor anti-apoptotics for preservation of neuronal function and preventionof neuronal death. Initial screens would be done in cell culture. Oneapplication would include prevention of neuronal death, by apoptosis, incerebral ischemia resulting from stroke. Apoptosis is known to beblocked by neuronal apoptosis inhibitory protein (NAIP); screens for itsupregulation, or effecting any coupled step could yield peptides whichselectively block neuronal apoptosis. Other applications includeneurodegenerative diseases such as Alzheimer's disease and Huntington'sdisease.

In a preferred embodiment, the present methods are useful in bonebiology applications. Osteoclasts are known to play a key role in boneremodeling by breaking down "old" bone, so that osteoblasts can lay down"new" bone. In osteoporosis one has an imbalance of this process.Osteoclast overactivity can be regulated by inserting candidatelibraries into these cells, and then looking for bioactive agents thatproduce: 1) a diminished processing of collagen by these cells; 2)decreased pit formation on bone chips; and 3) decreased release ofcalcium from bone fragments.

The present methods may also be used to screen for agonists of bonemorphogenic proteins, hormone mimetics to stimulate, regulate, orenhance new bone formation (in a manner similar to parathyroid hormoneand calcitonin, for example). These have use in osteoporosis, for poorlyhealing fractures, and to accelerate the rate of healing of newfractures. Furthermore, cell lines of connective tissue origin can betreated with candidate libraries and screened for their growth,proliferation, collagen stimulating activity, and/or prolineincorporating ability on the target osteoblasts. Alternatively,candidate libraries can be expressed directly in osteoblasts orchondrocytes and screened for increased production of collagen or bone.

In a preferred embodiment, the present methods are useful in skinbiology applications. Keratinocyte responses to a variety of stimuli mayresult in psoriasis, a proliferative change in these cells. Candidatelibraries can be inserted into cells removed from active psoriaticplaques, and bioactive agents isolated which decrease the rate of growthof these cells.

In a preferred embodiment, the present methods are useful in theregulation or inhibition of keloid formation (i.e. excessive scarring).Candidate libraries inserted into skin connective tissue cells isolatedfrom individuals with this condition, and bioactive agents isolated thatdecrease proliferation, collagen formation, or proline incorporation.Results from this work can be extended to treat the excessive scarringthat also occurs in burn patients. If a common peptide motif is found inthe context of the keloid work, then it can be used widely in a topicalmanner to diminish scarring post burn.

Similarly, wound healing for diabetic ulcers and other chronic "failureto heal" conditions in the skin and extremities can be regulated byproviding additional growth signals to cells which populate the skin anddermal layers. Growth factor mimetics may in fact be very useful forthis condition. Candidate libraries can be inserted into skin connectivetissue cells, and bioactive agents isolated which promote the growth ofthese cells under "harsh" conditions, such as low oxygen tension, lowpH, and the presence of inflammatory mediators.

Cosmeceutical applications of the present invention include the controlof melanin production in skin melanocytes. A naturally occurringpeptide, arbutin, is a tyrosine hydroxylase inhibitor, a key enzyme inthe synthesis of melanin. Candidate libraries can be inserted intomelanocytes and known stimuli that increase the synthesis of melaninapplied to the cells. Bioactive agents can be isolated that inhibit thesynthesis of melanin under these conditions.

In a preferred embodiment, the present methods are useful inendocrinology applications. The retroviral peptide library technologycan be applied broadly to any endocrine, growth factor, cytokine orchemokine network which involves a signaling peptide or protein thatacts in either an endocrine, paracrine or autocrine manner that binds ordimerizes a receptor and activates a signaling cascade that results in aknown phenotypic or functional outcome. The methods are applied so as toisolate a peptide which either mimics the desired hormone (i.e.,insulin, leptin, calcitonin, PDGF, EGF, EPO, GMCSF, IL1-17, mimetics) orinhibits its action by either blocking the release of the hormone,blocking its binding to a specific receptor or carrier protein (forexample, CRF binding protein), or inhibiting the intracellular responsesof the specific target cells to that hormone. Selection of peptideswhich increase the expression or release of hormones from the cellswhich normally produce them could have broad applications to conditionsof hormonal deficiency.

In a preferred embodiment, the present methods are useful in infectiousdisease applications. Viral latency (herpes viruses such as CMV, EBV,HBV, and other viruses such as HIV) and their reactivation are asignificant problem, particularly in immunosuppressed patients (patientswith AIDS and transplant patients). The ability to block thereactivation and spread of these viruses is an important goal. Celllines known to harbor or be susceptible to latent viral infection can beinfected with the specific virus, and then stimuli applied to thesecells which have been shown to lead to reactivation and viralreplication. This can be followed by measuring viral titers in themedium and scoring cells for phenotypic changes. Candidate libraries canthen be inserted into these cells under the above conditions, andpeptides isolated which block or diminish the growth and/or release ofthe virus. As with chemotherapeutics, these experiments can also be donewith drugs which are only partially effective towards this outcome, andbioactive agents isolated which enhance the virucidal effect of thesedrugs.

One example of many is the ability to block HIV-1 infection. HIV-1requires CD4 and a co-receptor which can be one of several seventransmembrane G-protein coupled receptors. In the case of the infectionof macrophages, CCR-5 is the required co-receptor, and there is strongevidence that a block on CCR-5 will result in resistance to HIV-1infection. There are two lines of evidence for this statement. First, itis known that the natural ligands for CCR-5, the CC chemokines RANTES,MIP1a and MIP1b are responsible for CD8+ mediated resistance to HIV.Second, individuals homozygous for a mutant allele of CCR-5 arecompletely resistant to HIV infection. Thus, an inhibitor of theCCR-5/HIV interaction would be of enormous interest to both biologistsand clinicians. The extracellular anchored constructs offer superb toolsfor such a discovery. Into the transmembrane, epitope tagged,glycine-serine tethered constructs (ssTM V G20 E TM), one can place arandom, cyclized peptide library of the general sequence CXXXXXXXXXXC orC-(X)_(n) -C (SEQ ID NO:50). Then one infects a cell line that expressesCCR-5 with retroviruses containing this library. Using an antibody toCCR-5 one can use FACS to sort desired cells based on the binding ofthis antibody to the receptor. All cells which do not bind the antibodywill be assumed contain inhibitors of this antibody binding site. Theseinhibitors, in the retroviral construct can be further assayed for theirability to inhibit HIV-1 entry.

Viruses are known to enter cells using specific receptors to bind tocells (for example, HIV uses CD4, coronavirus uses CD13, murine leukemiavirus uses transport protein, and measles virus usesCD44) and to fusewith cells (HIV uses chemokine receptor). Candidate libraries can beinserted into target cells known to be permissive to these viruses, andbioactive agents isolated which block the ability of these viruses tobind and fuse with specific target cells.

In a preferred embodiment, the present invention finds use withinfectious organisms. Intracellular organisms such as mycobacteria,listeria, salmonella, pneumocystis, yersinia, leishmania, T. cruzi, canpersist and replicate within cells, and become active inimmunosuppressed patients. There are currently drugs on the market andin development which are either only partially effective or ineffectiveagainst these organisms. Candidate libraries can be inserted intospecific cells infected with these organisms (pre- or post-infection),and bioactive agents selected which promote the intracellulardestruction of these organisms in a manner analogous to intracellular"antibiotic peptides" similar to magainins. In addition peptides can beselected which enhance the cidal properties of drugs already underinvestigation which have insufficient potency by themselves, but whencombined with a specific peptide from a candidate library, aredramatically more potent through a synergistic mechanism. Finally,bioactive agents can be isolated which alter the metabolism of theseintracellular organisms, in such a way as to terminate theirintracellular life cycle by inhibiting a key organismal event.

Antibiotic drugs that are widely used have certain dose dependent,tissue specific toxicities. For example renal toxicity is seen with theuse of gentamicin, tobramycin, and amphotericin; hepatotoxicity is seenwith the use of INH and rifampin; bone marrow toxicity is seen withchloramphenicol; and platelet toxicity is seen with ticarcillin, etc.These toxicities limit their use. Candidate libraries can be introducedinto the specific cell types where specific changes leading to cellulardamage or apoptosis by the antibiotics are produced, and bioactiveagents can be isolated that confer protection, when these cells aretreated with these specific antibiotics.

Furthermore, the present invention finds use in screening for bioactiveagents that block antibiotic transport mechanisms. The rapid secretionfrom the blood stream of certain antibiotics limits their usefulness.For example penicillins are rapidly secreted by certain transportmechanisms in the kidney and choroid plexus in the brain. Probenecid isknown to block this transport and increase serum and tissue levels.Candidate agents can be inserted into specific cells derived from kidneycells and cells of the choroid plexus known to have active transportmechanisms for antibiotics. Bioactive agents can then be isolated whichblock the active transport of specific antibiotics and thus extend theserum halflife of these drugs.

In a preferred embodiment, the present methods are useful in drugtoxicities and drug resistance applications. Drug toxicity is asignificant clinical problem. This may manifest itself as specifictissue or cell damage with the result that the drug's effectiveness islimited. Examples include myeloablation in high dose cancerchemotherapy, damage to epithelial cells lining the airway and gut, andhair loss. Specific examples include adriamycin induced cardiomyocytedeath, cisplatinin-induced kidney toxicity, vincristine-induced gutmotility disorders, and cyclosporin-induced kidney damage. Candidatelibraries can be introduced into specific cell types with characteristicdrug-induced phenotypic or functional responses, in the presence of thedrugs, and agents isolated which reverse or protect the specific celltype against the toxic changes when exposed to the drug. These effectsmay manifest as blocking the drug induced apoptosis of the cell ofinterest, thus initial screens will be for survival of the cells in thepresence of high levels of drugs or combinations of drugs used incombination chemotherapy.

Drug toxicity may be due to a specific metabolite produced in the liveror kidney which is highly toxic to specific cells, or due to druginteractions in the liver which block or enhance the metabolism of anadministered drug. Candidate libraries can be introduced into liver orkidney cells following the exposure of these cells to the drug known toproduce the toxic metabolite. Bioactive agents can be isolated whichalter how the liver or kidney cells metabolize the drug, and specificagents identified which prevent the generation of a specific toxicmetabolite. The generation of the metabolite can be followed by massspectrometry, and phenotypic changes can be assessed by microscopy. Sucha screen can also be done in cultured hepatocytes, cocultured withreadout cells which are specifically sensitive to the toxic metabolite.Applications include reversible (to limit toxicity) inhibitors ofenzymes involved in drug metabolism.

Multiple drug resistance, and hence tumor cell selection, outgrowth, andrelapse, leads to morbidity and mortality in cancer patients. Candidatelibraries can be introduced into tumor cell lines (primary and cultured)that have demonstrated specific or multiple drug resistance. Bioactiveagents can then be identified which confer drug sensitivity when thecells are exposed to the drug of interest, or to drugs used incombination chemotherapy. The readout can be the onset of apoptosis inthese cells, membrane permeability changes, the release of intracellularions and fluorescent markers. The cells in which multidrug resistanceinvolves membrane transporters can be preloaded with fluorescenttransporter substrates, and selection carried out for peptides whichblock the normal efflux of fluorescent drug from these cells. Candidatelibraries are particularly suited to screening for peptides whichreverse poorly characterized or recently discovered intracellularmechanisms of resistance or mechanisms for which few or nochemosensitizers currently exist, such as mechanisms involving LRP (lungresistance protein). This protein has been implicated in multidrugresistance in ovarian carcinoma, metastatic malignant melanoma, andacute myeloid leukemia. Particularly interesting examples includescreening for agents which reverse more than one important resistancemechanism in a single cell, which occurs in a subset of the most drugresistant cells, which are also important targets. Applications wouldinclude screening for peptide inhibitors of both MRP (multidrugresistance related protein) and LRP for treatment of resistant cells inmetastatic melanoma, for inhibitors of both p-glycoprotein and LRP inacute myeloid leukemia, and for inhibition (by any mechanism) of allthree proteins for treating pan-resistant cells.

In a preferred embodiment, the present methods are useful in improvingthe performance of existing or developmental drugs. First passmetabolism of orally administered drugs limits their oralbioavailability, and can result in diminished efficacy as well as theneed to administer more drug for a desired effect. Reversible inhibitorsof enzymes involved in first pass metabolism may thus be a usefuladjunct enhancing the efficacy of these drugs. First pass metabolismoccurs in the liver, thus inhibitors of the corresponding catabolicenzymes may enhance the effect of the cognate drugs. Reversibleinhibitors would be delivered at the same time as, or slightly before,the drug of interest. Screening of candidate libraries in hepatocytesfor inhibitors (by any mechanism, such as protein downregulation as wellas a direct inhibition of activity) of particularly problematicalisozymes would be of interest. These include the CYP3A4 isozymes ofcytochrome P450, which are involved in the first pass metabolism of theanti-HIV drugs saquinavir and indinavir. Other applications couldinclude reversible inhibitors of UDP-glucuronyltransferases,sulfotransferases, N-acetyltransferases, epoxide hydrolases, andglutathione S-transferases, depending on the drug. Screens would be donein cultured hepatocytes or liver microsomes, and could involveantibodies recognizing the specific modification performed in the liver,or cocultured readout cells, if the metabolite had a differentbioactivity than the untransformed drug. The enzymes modifying the drugwould not necessarily have to be known, if screening was for lack ofalteration of the drug.

In a preferred embodiment, the present methods are useful inimmunobiology, inflammation, and allergic response applications.Selective regulation of T lymphocyte responses is a desired goal inorder to modulate immune-mediated diseases in a specific manner.Candidate libraries can be introduced into specific T cell subsets (TH1,TH2, CD4+, CD8+, and others) and the responses which characterize thosesubsets (cytokine generation, cytotoxicity, proliferation in response toantigen being presented by a mononuclear leukocyte, and others) modifiedby members of the library. Agents can be selected which increase ordiminish the known T cell subset physiologic response. This approachwill be useful in any number of conditions, including: 1) autoimmunediseases where one wants to induce a tolerant state (select a peptidethat inhibits T cell subset from recognizing a self-antigen bearingcell); 2) allergic diseases where one wants to decrease the stimulationof IgE producing cells (select peptide which blocks release from T cellsubsets of specific B-cell stimulating cytokines which induce switch toIgE production); 3) in transplant patients where one wants to induceselective immunosuppression (select peptide that diminishesproliferative responses of host T cells to foreign antigens); 4) inlymphoproliferative states where one wants to inhibit the growth orsensitize a specific T cell tumor to chemotherapy and/or radiation; 5)in tumor surveillance where one wants to inhibit the killing ofcytotoxic T cells by Fas ligand bearing tumor cells; and 5) in T cellmediated inflammatory diseases such as Rheumatoid arthritis, Connectivetissue diseases (SLE), Multiple sclerosis, and inflammatory boweldisease, where one wants to inhibit the proliferation of disease-causingT cells (promote their selective apoptosis) and the resulting selectivedestruction of target tissues (cartilage, connective tissue,oligodendrocytes, gut endothelial cells, respectively).

Regulation of B cell responses will permit a more selective modulationof the type and amount of immunoglobulin made and secreted by specific Bcell subsets. Candidate libraries can be inserted into B cells andbioactive agents selected which inhibit the release and synthesis of aspecific immunoglobulin. This may be useful in autoimmune diseasescharacterized by the overproduction of auto antibodies and theproduction of allergy causing antibodies, such as IgE. Agents can alsobe identified which inhibit or enhance the binding of a specificimmunoglobulin subclass to a specific antigen either foreign of self.Finally, agents can be selected which inhibit the binding of a specificimmunoglobulin subclass to its receptor on specific cell types.

Similarly, agents which affect cytokine production may be selected,generally using two cell systems. For example, cytokine production frommacrophages, monocytes, etc. may be evaluated. Similarly, agents whichmimic cytokines, for example erythropoetin and IL1-17, may be selected,or agents that bind cytokines such as TNF-α, before they bind theirreceptor.

Antigen processing by mononuclear leukocytes (ML) is an important earlystep in the immune system's ability to recognize and eliminate foreignproteins. Candidate agents can be inserted into ML cell lines and agentsselected which alter the intracellular processing of foreign peptidesand sequence of the foreign peptide that is presented to T cells by MLson their cell surface in the context of Class II MHC. One can look formembers of the library that enhance immune responses of a particular Tcell subset (for example, the peptide would in fact work as a vaccine),or look for a library member that binds more tightly to MHC, thusdisplacing naturally occurring peptides, but nonetheless the agent wouldbe less immunogenic (less stimulatory to a specific T cell clone). Thisagent would in fact induce immune tolerance and/or diminish immuneresponses to foreign proteins. This approach could be used intransplantation, autoimmune diseases, and allergic diseases.

The release of inflammatory mediators (cytokines, leukotrienes,prostaglandins, platelet activating factor, histamine, neuropeptides,and other peptide and lipid mediators) is a key element in maintainingand amplifying aberrant immune responses. Candidate libraries can beinserted into MLs, mast cells, eosinophils, and other cellsparticipating in a specific inflammatory response, and bioactive agentsselected which inhibit the synthesis, release and binding to the cognatereceptor of each of these types of mediators.

In a preferred embodiment, the present methods are useful inbiotechnology applications. Candidate library expression in mammaliancells can also be considered for other pharmaceutical-relatedapplications, such as modification of protein expression, proteinfolding, or protein secretion. One such example would be in commercialproduction of protein pharmaceuticals in CHO or other cells. Candidatelibraries resulting in bioactive agents which select for an increasedcell growth rate (perhaps peptides mimicking growth factors or acting asagonists of growth factor signal transduction pathways), for pathogenresistance (see previous section), for lack of sialylation orglycosylation (by blocking glycotransferases or rerouting trafficking ofthe protein in the cell), for allowing growth on autoclaved media, orfor growth in serum free media, would all increase productivity anddecrease costs in the production of protein pharmaceuticals.

Random peptides displayed on the surface of circulating cells can beused as tools to identify organ, tissue, and cell specific peptidetargeting sequences. Any cell introduced into the bloodstream of ananimal expressing a library targeted to the cell surface can be selectedfor specific organ and tissue targeting. The bioactive agent sequenceidentified can then be coupled to an antibody, enzyme, drug, imagingagent or substance for which organ targeting is desired.

Other agents which may be selected using the present inventioninclude: 1) agents which block the activity of transcription factors,using cell lines with reporter genes; 2) agents which block theinteraction of two known proteins in cells, using the absence of normalcellular functions, the mammalian two hybrid system or fluorescenceresonance energy transfer mechanisms for detection; and 3) agents may beidentified by tethering a random peptide to a protein binding region toallow interactions with molecules sterically close, i.e. within asignalling pathway, to localize the effects to a functional area ofinterest.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1

Proof of Concept Experiments

A number of systems were used to prove that the retroviral constructsoutlined herein were able to result in a selectable phenotype.

BcI2 and CPP32 Protection from apoptosis

It is known that BcI2 and the CPP32 peptide is able to inhibit apoptosisinduced by tumor necrosis factor and cycloheximide.

Apotag assay: TUNEL (TdT-mediated dUTP-fluorescein nick end labeling)Boehringer Mannheim kit, catalog no. 168795

3T3 cells transiently infected with either MFGLacZ, BCL2, or CPP322plasmids were grown to 50% confluence at the time of induction withhTNFa (50 ng/ml media) and cycloheximide (100 mg/ml media) for 6 hours.Cells were washed at 6 hours and harvested at 24 hours after induction.Cells were harvested by pooling all media from cells (in order tocollect any apoptotic, floating cells) with the washings and trypsinizedcells. The cells were spun and washed with PBS containing 1% BSA,transferred to an eppendorf tube and the wash repeated once.

Cells were fixed in 4% paraformaldehyde at room temperature for 30minutes, washed in PBS/BSA, then resuspended in permeabilisation bufferfor 2 minutes on ice. After permeabilisation, cells were washed twice inPBS/BSA and incubated at 37° C. for 1 hour with labeling buffercontaining fluoresceinated dUTP, unlabeled nucleotide mixture andterminal deoxynucleotidyl transferase (TdT). Cells were washed twicewith PBS/BSA, resuspended in PBS/BSA and transferred to a FACS tube foranalysis. Samples were also visualized under the fluorescencemicroscope. The results showed that expression of BcI2 or the CPP32peptide in 3T3 cells from an MSCV retroviral promoter in vivo was ableto inhibit apoptosis induced by tumor necrosis factor and cycloheximide.

Propidium Iodide staining of fixed cells to ass for apoptosis: (Sherwoodand Schimke, Methods in Cell Biology, 46:77-87, 1995) 3T3 cellstransiently infected with MFGLacZ, Bcl2, or CPP32 were plated andtreated with TNF/CXH as described above, and harvested and washed asabove. Cells were then resuspended in 70% ethanol in PBS at 4° C. andkept at 4° C. overnight. When ready to FACS, cells were stained withpropidium iodide as follows. Cells were spun at 14,000 RPM for 10seconds and washed once with PBS/BSA. Cells were then resuspended in 50ml staining solution (PBS with 50 mg/ml RNase A (DNase-free) with 10mg/ml propidium iodide) and incubated at 37° C. for at least 1 hour.Cells were then pelleted and resuspended in PBS/BSA solution containing10 mg/ml propidium iodide and analyzed by FACS scanning. The resultsshowed that expression of BCL2 or CPP32 peptide in 3T3 cells was able toinhibit apoptosis induced by tumor necrosis factor and cycloheximide asmeasure by Pl staining of cells, extending our previous results.

Ethidium Bromide/Acridine Orange Staining of BAF3 Cells to Study CellMorphology:

BAF3 cells were infected with WZL IRES NEO retroviral vectors containingno insert (WIN) or DNA coding for LacZ (ZIN), Bcl2 (BIN), CPP32 peptide(CIN), or scrambled peptide control (PIN). Cells were selected in G418after infection with above retroviral vectors and survivors werestimulated with 5 mg/ml FAS antibody. After stimulation, cells werestained with ethidium bromide and acridine orange (2 mg/ml each) andvisualized under the fluorescence microscope using the ultravioletfilter. 250 cells were counted and the percent of cells which wereapoptotic were calculated. Similar to results obtained in 3T3 cellsstimulated with TNF/CXH, the CPP32 encoding vectors are able to inhibitFAS induced apoptosis. The peptide control also had an effect in thissystem approximately half of that seen with BCL2 or the CPP32 peptide.

Enzymatic Assay of CPP32 activity:

CPP32 Assay Kit: Clontech CPP32 Colorimetric Assay Kit (Cat. No.K2027-2): 3T3 cells were infected with the vectors described in Part I,section C, and selected in G418 media prior to assay. 6-well plates of3T3 cells at near confluence were stimulated with TNF/CXH as describedabove and harvested at 30 min, 1, 2 and 4 hours after stimulation asfollows. Cells were trypsinized and collected as described above. Aftertransfering to an eppendorf tube, cells were spun and resuspended in 50ml chilled Cell Lysis Buffer. Cells were incubated for 10 minutes onice, then 50 ml of 2× Reaction Buffer containing DTT was added to eachtube. 5 ml of the colorimetric conjugated substrate(DEVD-paranitroanilide, 50 mM final concentration) was added to eachtube and incubated at 37° C. for 30 minutes. Samples were transferred toa 96 well plate and read on a spectrophotometer at O.D. of 405. Theresults showed that cell extracts from WIN cells have increased CPP32enzyme activity at 2 hours as measured by cleavage of DEVD-pNA substrateto its colorometrically detectable form pNA. By 4 hours, cells havebegun to die and the activity is decreased. In cells containing BCL2 orthe CPP32 peptide inhibitor, this rise in activity is not seen. In thecase of BCL2, it should be due to inhibition of apoptosis upstream ofthe enzyme. With CPP32 inhibitor peptide, it should be due to directinhibition of enzymatic activity. These in vitro results are consistentwith the results seen in cell death assays described above.

Localization studies using PKC inhibitor

Murine 1OT1/2 Clone 8 cells were stimulated with PMA which is known tocause translocation of PKC from the cytoplasm to the nucleus. Thistranslocation is thought to be mediated through binding to a protein atthe site of action, termed a RAC (receptor for activated protein kinaseC) protein. Uninfected clone 8 cells were compared to cells infectedwith pBabe puro retroviral constructs containing sequences coding foreither Flu-epitope (MGGGYPYDVPDYAGSLZ) (SEQ ID NO:51) tagged scrambledpeptide control or inhibitor peptide (GKQKTKTIKGPP) (SEQ ID NO:52) whichis identical to the C2 region of all the PKC isozymes. We then assayedthe cells by immunohistochemistry using an antibody specific for PKCaand visualized with a secondary antibody conjugated to horseradishperoxidase.

This experiment was done at two different cell densities as follows:

1. Cells were plated at 2,000 cells/cm² onto 22 mm square polylysinecoated coverslips and allowed to grow for 2 days. On 3/20, cells werenearly confluent. Cells were replated at a lower density and assayedwith identical conditions on 3/27.

2. PMA was added at 10⁻⁵ M to the media for 30 minutes at 37° C.

3. Cells were rinsed with SCB buffer (physiologic buffer prewarmed to37° C. before use) and then placed into 3.7% glutaraldehyde in SCBbuffer for 20 minutes at 37° C.

4. Cells were then washed in SCB buffer then incubated with SCBT (SCBcontaining 0.1% Triton X-100) for 10 minutes at room temperature.

5. Coverslips were removed from the 6-well plate and dip washed in 0.1 %tween/PBS at room temperature and placed onto parafilm in a coveredcontainer.

6. Coverslips were incubated with 1.5% goat blocking serum in PBS withagitation in a humidified environment at room temperature.

7. Solutions were aspirated off the coverslip and coverslips were thenwashed with PBS. Primary anti-PKCa antibody was placed onto coverslipsand incubated for 30 minutes at room temperature as above. A 1:500dilution of antibody was used in all experiments.

8. Coverslips were then washed with PBS three times and then incubatedfor 30 minutes at room temperature with biotin-conjugated second stepantibody as provided in Santa Cruz ABC ImmunoStain Sytems kit.Coverslips were then washed three times with PBS.

9. Coverslips were then incubated in avidin biotin enzyme reagent (assupplied with kit) for 30 minutes at room temperature. Coverslips werethen washed for 10 minutes in PBS after being placed back into 6-wellplates.

10. Coverslips were rinsed with 0.5% Triton X-100/PBS for 30 seconds andincubated in DAB solution for 5 minutes. Reaction was stopped byaddition of distilled water to well.

11. Coverslips were then dehydrated through alcohols and xylene andmounted onto slides with Permount and visualised and photographed bylight microscopy.

The result showed that basically, control clone 8 cells showedpredominantly cytoplasmic and perinuclear staining, while PMA inducedcells consistently showed translocation to the nucleus. Cells infectedwith constructs coding for the scrambled peptide showed similarstaining. Cells infected with constructs coding for peptides identicalto the C2 region of PKC showed predominantly cytoplasmic and perinuclearstaining in both control and PMA induced cells suggesting that thispeptide is able to specifically inhibit translocation of activated PKCato its RAC protein upon stimulation of the cells with PMA. It is alsopossible using similarly infected cells to see the downstream results ofpeptide expression upon gene activity. Cells were infected withretroviruses expressing either the PKCb2.1, PKC2.1 peptide, a dominantnegative ras protein control, combinations of these viruses, or no virusat all. Cells were stimulated with PMA at 100 ng/ml, PDGF-AA, orPDGF-BB. mRNA was prepared and northern blots were performed for fosgene expression (induced by PKC activation) or the ribosomal protein P0,a loading control mRNA whose expression is not known to be acted upon bysignaling systems induced by PKC. The PKC peptides can markedly reduceexpression of the fos gene mRNA. Indeed, an unexpected result was thatunder certain conditions there is activation of the mRNA expression.This latter results confirms that novel outcomes can occur uponexpression of peptides within cells.

Example 2

pBabe Puro Retroviral Libraries and Apoptosis

A series of retroviral constructs have been designed for expression ofrandomized and biased peptides within target cell populations. Thepeptide is expressed from a retroviral promoter. The translation unithas several important components. Glycine following the initiatormethionine at the amino terminus stabilizes the peptide and enhancescytoplasmic half-life, according to Varshavsky's N-End Rule. In someconstructs, a nine amino acid flu epitope tag has been incorporated topermit co-precipitation of the rare peptide and any molecule to which ithas affinity, by using monoclonal antibodies to the epitope. Glycinesare encoded before and after the random/biased expression productencoding regions to provide some molecular flexibility. Twocarboxyl-terminal prolines are encoded to confer stability tocarboxypeptidase.

For construction of a large library two primers were made (schematizedin FIG. 1). The first, designated the random peptide primer, consists of1 ) a complementary region for vector priming, 2) the regions mentionedabove, and 3) a random or biased expression product region, werepresented as a 30 base sequence encoding a peptide of length 10 aminoacids. In addition, we have inserted a stop codon in all three readingframes in case of minor deletions or insertions in the random region.The design of the primer ensures a glycine/proline termination in mostreading frames. The second primer is downstream in the vector and primesa region of the plasmid that contains a unique Not I site. These primersare used to create a library of fragments, each containing a differentnucleotide sequence that each potentially encodes a different peptide.These families of fragments are ligated to vector fragments containingpuromycin selection sequence, a 3'LTR, and a bacterial origin ofreplication. The ligation products are then electroporated into E. coliand DNA is prepared from the resulting library. Using this technique, wehave constructed independent random libraries with up to 2×10⁸ uniqueinserts. Sequencing multiple individual inserts demonstrates they havethe structure as defined by Primer 1, and the peptides encoded arerandom. Such libraries thus made contain subsets of the total 10¹³predicted peptides.

Generation of Retroviral Peptide Libraries

A scheme for generating a peptide library in the pBabe Puro vector isshown in FIG. 2. Primers for PCR were synthesized, purified anddeprotected according to standard protocols. Primer 1, complementary topolylinker sequences in the pBabe Puro retroviral construct, has thesequence 5' GCT TAG CAA GAT CTC TAC GGT GGA CCK NNK NNK NNK NNK NNK NNKNNK NNK NNK NNC CCC ACT CCC ATG GTC CTA CGT ACC ACC ACA CTG GG 3' (SEQID NO:53). N represents any of the four bases; K is limited to G or T.Primer 2 has the sequence 5' GCT TAG CM GAT CTG TGT GTC AGT TAG GGT GTGG 3' (SEQ ID NO:54) and is complementary to sequences within the pUC1 8origin of replication. PCR was carried out for 8 rounds using primer 1,primer 2, Babe Puro as template, and a mixture of Taq DNA Polymerase(Promega) and Deep Vent DNA Polymerase (New England Biolabs) in a ratioof 128 Taq: 1 Deep Vent as described in Barnes (1994) Proc. Natl. Acad.Sci. USA, 91, pp. 2216-2220. The amplified PCR product was purified,digested with restriction enzymes Bgl II and Not I (Promega), purifiedagain and ligated with the corresponding Bam HI-Not I fragment of pBabePuro. After transformation the resulting library contained ˜2×10⁸clones, greater than 80% of which contained inserts.

pMSCV-PC and pBabeMN-PC retroviral construct libraries:

Oligonucleotides were synthesized and purified according to standardprotocols. The "library" oligonucleotides have the sequence 5' CTG GAGAAC CAG GAC CAT GGG C (NNK)10 GGG CCC CCT TM ACC ATT AAA T 3' (SEQ IDNO:55) or 5' CTG GAG MC CAG GAC CAT GGG CNN KNN KNN KCC TCC CNN KCC TNNKNN KGG GCC CCC TTA MC CAT TM AT 3' (SEQ ID NO:56). A thirdoligonucleotide ("constant"), complementary to the 3' ends of thelibrary oligonucleotides, has the sequence 5'TCA TGC ATC CM TTT MT GGTTTA AG 3' (SEQ ID NO:57). As shown in FIG. 3, each libraryoligonucleotide is annealed to the constant oligonucleotide, convertedto double stranded DNA with Sequenase (United States Biochemical) orKlenow (Promega), digested with restriction enzyme Bst Xl (New EnglandBiolabs), and purified and ligated with the appropriate Bst XI-digestedretroviral construct. Transformation efficiencies are ˜2×10⁸ clones permicrogram of ligated DNA, greater than 90% of which contain an insert. Arepresentative retrovirus is shown in FIG. 4; see also, retroviralnucleotide sequence below:

Retroviral vector with presentation construct.

TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTMCGCCATTTTGCAMGGCATGGMAATACATMCTGAGMTAGAGMGTTCAGATCMGGTTAGGAACAGAGAGACAGCAGMTATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCMGMCAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGT TTCTAGAGAACCATCAGATGTTCCAGGGTGCCCCAAGGACCTGAAMTGACCCTGTGCCTTATTTGAACTMCCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCMTAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTATTCCCAATMAGCCTCTTGCTGTTTGCATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCUTTCATTTGGAGGTTCCACCGAGATTTGGAGACCCCTGCCTAGGGACCACCGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCGTTTCCTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCGGCATCTMTGTTTGCGCCTGCGTCTGTACTAGTTAGCTMCTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCTGMCACCCGGCCGCMCCCTGGGAGACGTCCCAGGGACTTTGGGGGCCGTTTTTGTGGCCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTIIGGAACCGAAGCCGCGCGTCTTGTCTGCTGCAGCGCTGCAGCATCGTTCTGTGTTCTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATFAGGGCCAGACTGTTACCACTCCCTTAAGTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACMCCAGTCGGTAGATGTCAAGMGAGACGTTGGGTTACCTTCTGCTCTGCAGMTGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCGGMTTCCAGGACCATGGGCGGGCCCCCTTAMCCATTAATTGGTAMATAAAGGATCCGTCGACCTGCAGCCMGCTTATCGATAAAATAAAAGATTTTATTIAGTCTCCAGAAAAAGGGGGGMTGAAAGACCCCACCTGTAGGWTTGGCAAGCTAGCTTMGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGMTATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCMGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTiCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCMGGACCTGMAATGACCCTGTGCCTTATTTGMCTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCIIICATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACMTTCCACACMCATACGAGCCGGAAGCATAMGTGTAMGCCTGGGGTGCCTMTGAGTGAGCTAACTCACATTMTTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAMCCTGTCGTGCCAGCTGCATTMTGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGMAGAACATGTGAGCMAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGITTIICCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAMCCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGMGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGMGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGMCCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAMACTCACGTTAAGGGAITTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAMTTAAAAATGAAGTTTTIMATCAATCTAAAGTATATATGAGTAMCTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTAWTICGTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGMGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTMTTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCMCGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAMGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTMGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAMGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAMACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCMCTGATCTTCAGCATCTTTACTTTCACCAGCGTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAMGGGAATMGGGCGACACGGMATGTTGMTACTCATACTCTFCCTTTTTCAATATTATTGMGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAITIFAGAAAAATAMCAMTAGGGGTTCCGCGCACATTTCCCCGAAMGTGCCACCTGACGTCTMGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCMCTGTTGGGMGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCMGGCGATTMGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAMACGACGGCCAGTGCCACGCTCTCCCTTATGCGACTCCTGCATTAGGMGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCMCAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAG (SEQ ID NO:58)

Peptide Library Infection of a Factor-dependent Line and Outgrowth of anApoptosis-Resistant Line.

The Baf/3 cell line is an IL-3 dependent cell that undergoes rapidapoptosis in the absence af IL-3. Thus it makes an attractive cell linefor dominant effector peptides.

Cells expressing a peptide that inhibits apoptosis are readily selectedagainst the background of dying cells. We chose this cell line as amodel for demonstrating peptide selection.

A retroviral library containing 5×10⁵ independent peptide inserts wastransfected into BOSC23 cells and converted into retrovirus with anappropriate titer of 5×10⁵ per ml. Twelve ml of viral supernatant wasused to infect 6×10⁶ Baf/3cells (2 ml [per infection of 1×10⁶ cells inindependent infections). Cells were grown for 3 days after infection inthe presence of IL-3 to allow retroviral integration and peptideexpression. After three days IL-3 was withdrawn and the cells allowed togrow for two weeks. After two weeks, one well of six had outgrowth ofcells that survive in the absence of IL-3, indicating the presence of anapoptosis-inhibiting peptide. Peptides derived in this manner may effectthe IL-3 independence by positive dominancy (i.e., mimic or circumventthe positive regulatory role of IL-3) or by inhibition (i.e., preventthe apoptosis process upon IL-3 withdrawal).

Example 3

pMSCVpc Vector Construction and Apoptosis

The retroviral vector pMSCVpc was prepared by cloning an insertcontaining sequences encoding a Kozak translation initiation sequence,BstXI sites for cloning library inserts, NruI and XhoI sites and stopcodons in all three reading frames, into the EcoRI and BamHI sites ofpMSCV neo.

BstX I Restriction Digestion

200 μg pMSCVpc vector DNA was combined with 40 μl 10X NEBuffer 3 and 30μl BstX I (10 units/μl) in a total volume of 400 μl. The sample wasincubated overnight at 55° C., phenol extracted, and digested with XhoI,and purified on a potassium acetate step gradient using 10, 15, 20 and25% solutions of potassium acetate. The DNA was PreciPitated, with arecovery of 40%.

Library Insert Preparation

Oligonucleotide Synthesis

Oligonucleotides (OL) with the following sequences were synthesized:OL-1: 5'-CTG GAG MC CAG GAC CAT GGG CM GAG AAA GGG CGA TGA GGT GGA TGGAGT GGG GCC CCC TTA MC CAT TM AT 3' (SEQ ID NO:59). The underlinedregion encodes a peptide with the sequence MGKRKGDEVDGVGPP (SEQ IDNO:60). This peptide was shown to inhibit Fas-mediated and Staurosporininduced apoptosis when expressed in cells with a retrovirus. OL-2:5'-CTG GAG AAC CAG GAC CAT GGG CM GAG AAA GGG CNN KNN KNN KGA KNN KGTGGG GCC CCC TTA MC CAT TM AT-3' (SEQ ID NO:61) Variable region: N=A, C,G, T (equimolar) K=G, T (equimolar) Limiting the K position of eachcodon to G or T reduces stop codon generation and codon usage bias. Theunderlined region encodes a randomized peptide with the sequenceMGKRKGXXXD/EXVGPPA (SEQ ID NO:62).

OL-3: 5'-TCA TGC ATC CAA TTT AAT GGT TTA AG-3' (SEQ ID NO:63) The 153'-bases of OL-3 are complementary to the 15 3'-bases of OL-1 and OL-2.

OL-1 and OL-2 were synthesized at 1 μM scale, while OL-3 was synthesizedat standard 40 nM scale. All of the oligos were synthesized withtrityl-on, deprotected and purified on OPC columns according to themanufacturer's directions (Applied Biosystems). Each oligo wasresuspended in 200 μl 10 mM Tris pH 8.5 without EDTA. The DNAconcentration was determined by measuring the absorbance at 260 nm.

PCR was done with 50 pmole of either OL-1 or OL-2 and 50 pmole of OL-3.Phenol extraction and ethanol precipitation was done, and the resultingDNA was run on a 10% nondenaturing 10% acrylamide gel, with ethidiumbromide staining.

The samples were quantitated, ligated, precipitated and electroporatedinto electrocompetent TOP10F' E. Coli (Invitrogen) using standardtechniques (see Current Protocols in Molecular Biology, section 1.8.4).A test transformation yielded 5×10⁹ transformants per μg of pUC DNA.After transformation, the transformation efficiency was determined byplating dilutions onto LB-amp plates (100 pg/ml ampicillin) and countingsurviving colonies. For the library insert generated from OL-2, a 4:1insert:vector molar ration in the ligation gave a transformationefficiency of 3.98×10⁷ transformants per pg vector DNA used in theligation, with a large scale transformation efficiency of 4.8×10⁷transformants per ug vector. The vector alone ligation generated 40 foldfewer transformants. 10 colonies from the transformation with the OL-1insert ligation were picked, cultured and the DNA prepared and sequencedto identify the correct clone.

The remainder of the OL-2 library SOC/transformation mixture wasinoculated into 500 μl LB-amp (100 μg/ml ampicillin) and incubated at37° C. with shaking (300 rpm). The Abs₆₀₀ of the library culture wasmonitored. When the culture reached an Abs₆₀₀ of 0.8 (approximately fivehours), 100 μl were removed, pelleted, resuspended in 10 ml LB/15%glycerol and stored in 1 ml aliquots at -80° C. (An Abs₆₀₀ of 0.8 equalsa cell concentration of approximately 10⁹ cells per ml. Therefore, for alibrary of 4.8×10⁷, each frozen aliquot will contain 200 libraryequivalents).

Analysis of library diversity

Surviving colonies plated above were screened by PCR with primersflanking the degenerate region to determine the fraction of clones whichcontained insert (>90%). 8 insert-containing clones were picked and thenucleotide sequences of the degenerate and flanking non-degenerateregions determined. Each nucleotide was represented in the N positionswith approximately 25% frequency, while G or T (but not A or C) wasrepresented in the K positions with approximately 50% frequency. Thefrequency of stop codons generated in the degenerate region can bedetermined by this method as well.

Generation of library retrovirus and infection of Jurkat cells.

DAY 0: Preparation of Phoenix Retrovirus Producer cells forTransfection: 18-24 hours prior to transfection, Phoenix cells wereevenly plated at 1.5-2 million cells per 60 mm plate in Producer cellgrowth media (DMEM: 10% FCS, 1% Penicillin-Streptomycin, 1% Glutamine).Cells were allowed to attach for 20 hours on the plates.

DAY 1: Transient transfection: The highest transfection frequencies areobtained with

Phoenix cells that are 70-80% confluent at the time of transfection. TheDNA in HBS (2×HBS=8.0 g NaCl, 6.5 g HEPES, 10 ml Na₂ HPO₄ stock (5.25 gdibasic in 500 ml water), adjusted to pH 7, to a final volume of 500mls, with a final pH adjustment to 7) was prepared for application tothe Phoenix cells. About 5 minutes prior to transfection, chloroquine(Sigma) was added to each plate to 25 uM (chloroquine stock is 50 mM inddH2O; for 3 mL media+1 ml DNA, add 2 μl). To a 15 ml conical tube, thefollowing were added (per 6 cm plate, 5 plates total, with all reagentsat room temperature):

5 ug library DNA (DNA was added in a drop to side of tube)

1 ug pMSCVpc lacZ virus vector

438 u1 dd H2O (the DNA was washed to the bottom of tube with water).

61 ul 2M CaC1₂ (Mallinkrodt, catalog #4160; make up in water, sterilefilter and store tightly capped at 4° C.

500 ul Total volume.

Samples were mixed thoroughly with finger tapping. Transfections with 5ug pMSCVpc lac Z and with the OL-1 vector DNA were carried out for useas negative and positive controls, respectively. 0.5 mL 2×HBS was addedto each tube quickly; the solution was bubbled vigorously with theautomatic pipettor by keeping the eject button depressed) for 10 sec(the actual length of bubbling time depends on each batch of 2×HBS). TheHBS/DNA solution was dispersed dropwise and evenly onto the media ineach Phoenix cell plate dropwise (gently and quickly). The plates wereobserved under a microscope; evenly distributed very small blackparticles of precipitated DNA (like pepper) were visible. The plateswere placed in a 37° C. incubator and rocked forward and backward a fewtimes to evenly distribute the DNA/CaPO4 particles. 6-8 hourspost-transfection, the media was changed to 3 ml fresh DMEM, 10% FCS.Prior to the media change, the DNA precipitate was larger and moreclearly visible under the microscope.

DAY 2: Second media change.

24 hours post-transfection, the media was changed again to 3 ml freshDMEM, 10% FCS. The cells were placed at 32° C. (the virus is more stableif incubation is carried out at 32° C., although 37° C. is fine).

DAY 3: Transduction of Jurkat EcoR cells.

A sterile Acrodisc 0.45 micron syringe filter (Gelman Sciences) wasattached to the end of a 10 ml sterile syringe and the injection stoppersterilely removed from the syringe barrel. At 48 hourspost-transfection, the virus supernatant was removed from the Phoenixcells and added to the syringe barrel. The stopper was replaced and thevirus supernatant was ejected dropwise into a clean, sterile conicaltube. The Phoenix cell plates were set aside for X-Gal staining (seebelow). Polybrene was added to each viral supernatant (Sigma; 2.5 mg/mlin ddH2O=500×; store at -20° C.) to a final concentration of 5 mg/ml.4.5×10⁶ Jurkat EcoR cells (Jurkat cells stably expressing the ecotropicretrovirus receptor) were pelleted for 1400 rpm for five min andresuspended in 9 mls of the OL-2 library virus supernatant. The cellswere distributed in aliquots of 1 ml, or 5×10⁵ cells, into the wells ofa 24 well plate. 1.5×10⁶ Jurkat EcoR cells were similarly treated with 3mls each of the lacZ viral supernatant and the OL-1 viral supernatant.Each cell plate was wrapped in parafilm, placed in a microplate carrier(DuPont) and centrifuged at 2500 rpm for 90 min at 32° C. in aDuPont/Sorvall RT 6000B table top centrifuge. After centrifugation, thecells were observed under a microscope. The presence of largeirregularly-shaped bodies representing fused Jurkats (each as large as5-10 unfused cells) suggested successful infection. The parafilm wasremoved from the plates, which were placed at 32° C. After an additional16 hours at 32° C., the cells were loosened from the bottom of each wellwith gentle trituration and added to a 15 ml conical tube. The tubeswere centrifuged at 1400 rpm for five min to the pellet the cells. Thecells were resuspended in 5 mls fresh RPMI ,10% FCS for every threewells of cells and added to a 60 mm plate (3 wells of cells per plate).1 ml fresh RPMI, 10% FCS was added to each well of cells remaining inthe 24 well plates. Plates were kept at 37° C. for 72 hours, at whichtime the cells transduced with each virus were combined and an aliquotJurkat cells stained with X-Gal. Unused viral supernatant was stored at-80° C. for future transduction, although the titer drops by one-halffor each freeze-thaw cycle.

Determination of transfection efficiency.

Both tranfected Phoenix cells and transduced Jurkat cells were stainedwith X-Gal to gauge the transfection and transduction efficiencies. Thepurpose of co-transfecting the pMSCVpc lacZ virus vector with thelibrary virus vector, as described above, was to permit an indirectassessment of the efficiencies of transfection and transduction.Preparation of solutions: fixative: PBS/0.10% Glutaraldehyde.Glutaraldehyde stock (Sigma cat #G5882) is a 25% solution, or 250×;stock staining solutions: i) 300 mM/25× ferrocyanate solution: 25.3 gK4Fe(CN)6.3H2O (Mallinckrodt)+2.48 g MgCl2 (Sigma) in 200 ml H2O; storeat 4° C.; ii. 300 mM/25× ferricyanate solution: 19.75 g K3Fe(CN)6(Sigma)+2.48 g MgCl2 in 200 ml H2O; store at 4°; iii. XGal (MolecularProbes) is made up as a 40 mg/ml solution in DMF; store at -20° C. inthe dark; iv. 1× ferro/ferricyanate solution: add 4 ml 300 mM/25×ferrocyanate solution and 4 ml 300 mM/25× ferricyanate solution to 196ml PBS; store at 4° C. for up to one month; v. active staining solution:each time cells are to be stained, 100 μl 40 mg/ml X-Gal is added toeach 3 ml 1× ferro/ferricyanate solution; washing solution: PBS forPhoenix and other adherent cells; 1% FCS in PBS for Jurkat and othernonadherent cells.

The media was removed from the 60 mm plates of Phoenix cells or 5×10⁵Jurkat cells were pelleted in a 15 ml conical tube at 1400 rpm for fivemin. 2 ml of fixative were added to each 60 mm plate of Phoenix cells orJurkat cells were resuspended in 1 ml fixative. Cells were left infixative for 2 min. For Phoenix cells, fixative was poured off and thecells were washed three times with PBS (first two washes were quick; forthird wash, the PBS was left on the cells for 3 min). For Jurkat cells,the fixative was quenched by adding 5-IO ml PBS/1% FCS to each conicaltube, inverting each tube five times and pelleting as before.3 ml ofactive staining solution were layered onto each 60 mm plate of Phoenixcells or each cell pellet of 5×10⁵ Jurkat cells was resuspended in 1 mlof active staining solution and placed in a well of a 24 well plate. Allcells were incubated at 37° C. The cells were observed under amicroscope 24 hours later. The efficiency of transfection of the Phoenixcells was estimated as the percentage of blue cells in a field. Theefficiency of transduction of the Jurkat cells was estimated by countingblue cells in a hemocytometer. Transfection with 5 μg lacZ vectorproduced 50% blue Phoenix cells. Transduction of Jurkats with theresulting virus produced 30% blue Jurkat cells. Co-transfection of 1 μglacZ virus vector with 5 μg library virus vector produced 5-10% bluePhoenix cells. Transduction of Jurkats with the resulting virus resultedin 3-10% blue Jurkat cells.

Selection of Jurkat cells with IgM anti-Fas.

Titer IgM anti-Fas: A fresh batch of CH-11 IgM antibody to human Fas(Kamiya Biomedical Company; cat #MC-060)was tested to determine theeffectiveness of induction of apoptosis. 5×10⁵ Jurkat EcoR cells werepelleted at 1500 rpm for five min and resuspended in 1 ml RPMI/2.5% FCSplus serial dilutions of CH-11 antibody, 50 ng/ml, 10 ng/ml, 2.0 ng/mland 0.5 ng/ml final concentration. Cells in each dilution of antibodywere placed in a well of a 24 well plate at 37° C. for 48 hours, atwhich time 4 ml acridine orange/ethidium bromide (Sigma; 100 μg/ml eachin PBS; store in the dark at 4° C.) was added to 100 ml cells on ice.Cells were examined in a hemocytometer under a 20× objective with afilter combination suitable for reading fluorescein.

2. 100 cells from each sample were counted and the number of cells inthe following groups was recorded:

1. live cells with normal nuclei (bright green chromatin with organizedstructure).

2. early apoptotic (EA; bright green chromatin that is highly condensedor fragmented).

3. late apoptotic (LA; bright orange chromatin that is highly condensedor fragmented).

4. necrotic cells (N; bright orange chromatin with organized structure).% apoptotic cells was calculated as EA+LA/total number of cellscounted×100 Using 10 ng/ml of the CH-11 antibody, >95% apoptosis ofJurkat EcoR cells was demonstrated.

IgM anti-Fas selection of library-expressing Jurkats.

9.6×10⁶ OL-2 library-transduced Jurkat cells were pelleted andresuspended in 96 ml RPMI/2.5% FCS+10 ng/ml CH-11 antibody. Cells weredistributed in 1 ml aliquots of 1×10⁵ cells into each well of four 24well plates. 4.8×10⁶ lacZ-transduced Jurkats and OL-1-transduced Jurkatswere similarly treated and each distributed into the wells of two 24well plates. Plates were placed at 37° C. for five days. The plates werechecked daily for bacterial or yeast contamination. Cells were removedfrom any contaminated wells and 2 ml 10N NaOH was added to the emptywells to reduce the risk of spread of contamination to other wells.Little to no live cells were observed under the microscope after 2-3days, confirmed by the red color of the media which had not beendepleted of any nutrients. Five days after initial IgM anti-Fastreatment, 1 ml RPMI/20% FCS was added to each well. The cells were leftat 37° C. for an additional 10-14 days. The plates were checkedfrequently for contamination and treated as above. 10 days afteraddition of the RPMI/20% FCS, nearly every well of the OL-1-transducedcells contained live colonies of cells, confirmed by the orange color ofthe nutrient-depleted media. The media in all wells of lacZ-transducedcells remained red, and little cell growth was observed in any of thewells. Selected wells of the OL-2 library-transduced cells containedlive cells and nutrient-depleted media. During the next two weeks, cellswere removed from all wells in which significant cell growth wasoccurring, as gaged by observing the cells directly under the microscopeand monitoring the increasing nutrient depletion of the cell media.Cells from each well were resuspended in 5 ml fresh RPMI/10% FCS andplaced in a 60 mm dish at 37° C. for 2-3 days.

RNA isolation

RNA was isolated from the each surviving well population of OL-2library-transduced Jurkat cells (17 wells), as well from five survivingwell populations of OL-1-transduced cells, using the mRNA Capture Kitaccording to the manufacturer's protocol (Boehringer-Mannheim cat#1 787896). Briefly: 5×10⁵ cells from each dish were pelleted at 1400 rpm forfive min in an Eppendorf tube, washed twice with PBS and resuspended in200 ml lysis buffer and sheared by passing six times through a 21 guageneedle attached to a 1 ml syringe. 4 ml 1:20 dilution of biotinylatedoligo(dT)20 was added to each sample and incubated for 3 min at 37° C.The mix was removed from each tube. Each tube was washed three timeswith 200 ml of washing buffer. Cells were also stored in 90% FCS/10%DMSO in 1 ml aliquots of 1×106 cells each in liquid nitrogen.

RT PCR rescue of peptide-encoding inserts from selected cells.

PCR was carried out using the Titan™ RT-PCR System (Boehringer Mannheimcat #1 855 476), using two primers: 5'pBL primer has the sequence:5'-GAT CCT CCC TTT ATC CAG-3' (SEQ ID NO:64) and is complementary tonucleotides 1364-1381 of all pMSCVpc-based vectors and retrovirus mRNA,just upstream of the cloned insert. 3A primer has the sequence 5'-CTACAG GTG GGG TCT TTC-3' (SEQ ID NO:65) and is complementary to a sequencein all pMSCVpc-based vectors and retrovirus mRNA, just downstream of thecloned insert.

Re-cloning rescued peptide-encoding inserts.

Each PCR-rescued sample was extracted with phenol chloroform, ethanolprecipitated and resupsended in 25 ml 10 mM Tris pH 8.5. 3 mlnondenaturing DNA gel loading dye was added to 10 ml of each sample andrun on a 10% acrylamide minigel with oligonucleotide quantitationstandards and a 10 base pair ladder, as described above. Each lanecontained one prominent band with the expected molecular weight of 216base pairs and minor background bands. The molarity of each sample wasquantitated using NIH Image as before. Each sample was BstXI restrictiondigested, phenol extracted, ethanol precipitated and resuspended in 25ml 10 mM tris pH 8.5.The purified samples were loaded onto 10%acrylamide gels and quantitated as before. All samples contained aprominent band of 55 base pairs, the expected molecular weight for therestriction digested insert, as well as bands of 100 base pairs and 51base pairs corresponding to each of the ends of the rescued DNA insertremoved by the restriction enzyme. Each restriction digested,PCR-rescued insert was ligated at a 4:1 insert:vector molar ratio with100 ng pMSCVpc vector DNA, precipitated and electrotransformed asbefore. Surviving colonies for each transformation were PCR screenedusing the 5'pBL and 3A primers. 8 to 10 insert-containing colonies foreach transformation were cultured overnight, the cultures were pooledand a single mini-DNA preparation carried out for each pool.

Fas-Selected Peptide clones: All peptides have the sequence: MET GLY LYSARG LYS GLY XXX XXX XXX D/E XXX VAL GLY PRO PRO (SEQ ID NO:62). Only thexxx xxx xxx D/E xxx (SEQ ID NO:100) amino acids are written above eachDNA sequence below.

From first library selection well:

L1B3 INDIVIDUAL CLONES, FAS-SELECTED.

(SEQ ID NO:67) THR ALA SER ASP ALA L1B3E1 ATG GGC AAG AGA AAG GGC ACGGCG TCT GAT GCT GTG GGG CCC CCT TAA (SEQ ID NO:66)

(SEQ ID NO:69) TYR PRO SER ASP VAL L1B3E2 ATG GGC AAG AGA AAG GGC TATCCT TCT GAT GTG GTG GGG CCC CCT TAA (SEQ ID NO:68)

(SEQ ID NO:71) THR PRO SER ASP MET L1B3E3 ATG GGC AAG AGA AAG GGC ACGCCT TCG GAT ATG GTG GGG CCC CCT TAA (SEQ ID NO:70)

(SEQ ID NO:73) THR ALA SER ASP LEU L1B3E6 ATG GGC AAG AGA AAG GGC ACGGCT TCT GAT CTT GTG GGG CCC CCT TAA (SEQ ID NO:72)

(SEQ ID NO:75) SER ASP ARG ASP ILE L1B3E7 ATG GGC AAG AGA AAG GGC TCTGAT AGG GAT ATT GTG GGG CCC CCT TAA (SEQ ID NO:74)

From second library selection well:

L2A5 INDIVIDUAL CLONES, FAS SELECTED.

(SEQ ID NO:77) TRP LEU LEU GLU PHE L2A5A2 ATG GGC AAG AGA AAG GGC TGGTTG CTA GAG TTT GTG GGG CCC CCT TAA (SEQ ID NO:76)

(SEQ ID NO:79) TRP LEU ILE GLU PHE L2A5A3 ATG GGC AAG AGA AAG GGC TGGTTG ATA GAG TTT GTG GGG CCC CCT TAA (SEQ ID NO:78)

(SEQ ID NO:77) TRP LEU LEU GLU PHE L2A5A6 ATG GGC AAG AGA AAG GGC TGGTTG CTA GAG TTT GTG GGG CCC CCT TAA (SEQ ID NO:76)

(SEQ ID NO:77) TRP LEU LEU GLU PHE L2A5A8 ATG GGC AAG AGA AAG GGC TGGTTG CTA GAG TTT GTG GGG CCC CCT TAA (SEQ ID NO:76)

(SEQ ID NO:81) SER TYR GLN ASP LEU L2A5A9 ATG GGC AAG AGA AAA GGC TCTTAC CAA GAT CTG GTG GGG CCC CCT TAA (SEQ ID NO:101)

EXAMPLE 3

Staurosporine selection of NIH 3T3 cells transduced with pBabe puropeptide library

A. Library construction. Construction of the pBabe puro random peptidelibrary was described earlier in the patent. The randomized peptide hasthe sequence: MGXXXXXXXXXXGGPP (SEQ ID NO:82). The diversity of thelibrary is 2×10⁸ at the DNA insert level.

B. Library transfection. Transfections were carried out as described forFas-selection, but in 15 cm plates of 10⁷ Phoenix cells. The DNAsolution added to each plate consisted of: 50 ug library DNA, 5 ug lacZvector, 4340 ul ddH₂ O, 610 ul 2M CaCl₂ and 5000 ul 2×HBS.

C. Library transduction. 24 hours prior to transduction, 2×10⁷ NIH 3T3cells were plated in each of ten 15 cm plates in 25 ml DMEM, 10% BovineCalf Serum. 5 ml library virus supernatant was added to each plate (pluspolybrene as before). 24 hour after transduction, media was changed to25 ml fresh DMEM, 10% BCS. Cells were stained with X-gal at 48 hourspost-transduction. The transduction efficiency was estimated as 40-50%.

D. Staurosporine selection. Staurosporine, an alkaloid from Streptomycessp., is a potent, broad spectrum inhibitor of protein kinases whichbinds the ATP site. Addition of 1 uM staurosporine in serum-free mediato NIH 3T3 cells induced >99% apoptosis within 24 hours, as determinedby ethidium bromide/acridine orange double staining as described for theFas selection.

2×10⁶ library-transduced NIH 3T3 cells were plated in each of 10 15 cmplates. Cells were allowed to attach for 24 hours, at which timestaurosporine was added to 1 uM in serum free DMEM. LacZ-transduced NIH3T3 cells and BCL-2-transduced NIH 3T3 cells were used as negative andpositive controls, respectively. 24 hours after staurosporine treatment,the media was changed to 25 ml fresh DMEM, 10% BCS. The media waschanged every two days for one week, until the surviving cells lookedhealthy (typical 3T3 morphology), at which time 1 uM staurosporine inserum-free media was added again. The media wash changed to DMEM, 10%BCS as before. Stp treatment was carried out again for a total of threetreatments, at which time the number of library-transduced cellssurviving appeared greater than the number of lacZ-transduced cells (butless than the BCL-2-transduced cells.

E. Moloney transfer. After the second staurosporine treatment, aliquotsof surviving cells from each plate were infected with wild type Moloneymurine leukemia virus supernatant. (generated by transfecting Phoenixcells with the retroviral vector pZap). The virus was allowed to spreadthrough the culture for one week (with re-plating of the cells every 2-3days). Cells were plated as before and treated with Staurosporine beforeproceeding to RNA isolation and PCR rescue.

F. RNA isolation. Aliquots of cells surviving in each plate wereresuspended in 90% FCS, 10% DMSO and stored in liquid nitrogen. RNA wasprepared with Trizol reagent (Gibco BRL, cat#15596-026). Briefly, 1 mlTRIzol reagent was added were 10 cm² monolayer of cells and incubatedfor 5 min at room temperature. Cell lysates were transferred to 15 mlconical tubes. (Note: at this point, DEPC-treated solutions andglassware were used exclusively). 0.2 ml chloroform was added per 1 mlTRIzol reagent used. Tubes were shaken for 15 sec, incubated for 3 minat room temperature and centrifuged at 12000×g for 15 min at 4° C. TheRNA-containing upper aqueous phase was removed and 0.5 ml isopropanoladded per 1 ml TRIzol used for the initial homogenization. Samples weremixed and incubated at room temperature for 10 min followed bycentrifugation as before. the supernatant was removed and the RNA pelletwashed with 75% ethanol (1 ml per 1 ml TRIzol). The sample was vortexedand centrifuged at 7500×g for 5 min at 4° C. The RNA pellet wasair-dried for 10 min and resuspended in RNase-free water with 10 minincubation at 60° C. to dissolve the pellet. RNA concentration wasdetermined by measuring the absorbance at 260 nm.

G. PCR rescue. PCR rescue was carried out as for Fas selection, usingthe primers 5'pBL and SV 40 down. The second primer has the sequence: 5'CTG ACA CAC ATT CCA CAG 3' (SEQ ID NO:83) and is complimentary topositions 1424-1441 of the pBabe Puro retroviral vector. PCR reactionswere extracted with phenol-chloroform, precipitated with ethanol anddigested with Bam HI and Sal I before ligation with the retroviralvector pWZL neo. The figure shows a 10% acrylamide gel of representativePCR-generated inserts:

Lane 1: 10 base pair ladder

Lane 2: undigested PCR insert from Stuarosporine-selected cellpopulation

Lane 3: undigested PCR insert from same cell population, after Moloneyrescue and Staurosporine selection.

Lanes 4 and 5: same as lanes 2 and 3, after restriction digestion.

H. Secondary screen. pWZI neo vectors containing rescued inserts weretransfected into Phoenix cells, and the resulting virus used totransduce NIH 3T3 cells. Staurosporine selection was repeated threetimes as before, before RNA preparation and PCR rescue.

I. Sequences of the first 9 positives:

The sequences of the first nine positives are as follows:

SEQUENCE OF 2 P 1

GGATCCAGTGTGGTGGTACGTAGGAATACC-ATG GGA TGT CCG TCT GTT GCT AGG CCG CGGGGT GGT GGG GGC CCC CCC Met Gly Cys Pro Ser Val Ala Arg Pro Arg Gly GlyGly Gly Pro Pro TAGCTAACTAAAGATCCCAGTGTGGTGGTACGTAGGAATTCGCC 2P1 (SEQ IDNO:84) Stp Stp Stp Bam/Bg (SEQ ID NO:85)

SEQUENCE OF 4 P 1

GGATCCCAGTGTGGTGGTACGTAGGAATACC-ATG GGA TTG TCT TTT GTT ATT (C/TGT CTGCAG CAT CGT GGG GGC CCC Met Gly Leu Ser Phe Val Ile Arg Leu Gln His ArgGly Gly Pro CCC TAG CTAACTAAAGATCCCAGTGTGGTGGTACGT 4P1 (SEQ ID NO:86)Pro Stp Stp StpBam/Bg (SEQ ID NO:87) Cys

SEQUENCE OF 5 P 1

GGATCCCAGTGTGGTGGTACGTAGGAGTACC-ATG GGA CCT CCG ATT TGG TAT ACT CAT TGGAGT CAT GGG GGC CCC CCC Met Gly Pro Pro Ile Trp Tyr Thr His Trp Ser HisGly Gly Pro Pro TAG CTAACTAAAGAT CC 5P1 (SEQ ID NO:88) Stp Stp StpBam/Bg(SEQ ID NO:89)

SEQUENCE OF 6 P 1

GGATCCCAGTGTGGTGGTACGTAGGAGTACC-ATG GAA GTC AGG CGT TTG TGA ATA CTC GGCATA AG GGG GGC CCC CCC Met Glu Val Arg Arg Leu Stp Gly Gly Pro Pro (SEQID NO:91) (SEQ ID NO:3) TAGCTAACTAAAGAT CC 6P1 (SEQ ID NO:90) Stp StpStpBam/Bg

SEQUENCE OF 7 P 1

CCGGCCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCC 7P1 (SEQID NO:92)

SEQUENCE OF 8 P 1

GGATCCCAGTGTGGTGGTACGTAGGAATACC ATG GGA CTT TAG CCG GGC CCCCCCTAGCTAACTAAAGATCCCAGTGTGGTGGT Met Gly Leu Stp Pro Pro Stp Stp StpBam/Bg ACGTAGGAATTCGCCAGCACAG T 8P1 (SEQ ID NO:93)

SEQUENCE OF 9 P 1

GGATCCCAGTGTGGTGGTACGTAGGAATAC ATG GGA ACT GTT ATG GCG ATG TCG GAT TAGGTC GAG GGG GGC CCC CCC Met Gly Thr Val Met Ala Met Ser Asp Stp Gly GlyPro Pro (SEQ ID NO:95) (SEQ ID NO:3) TAGCTAACTAAAGATCC 9P1 (SEQ IDNO:94) Stp Stp Stp Bam/Bg

SEQUENCE OF 10 P 1

GGATCCAGTGTGGTGGTACGTAGGAATACC ATG GGA TGT CCG TCT GTT GCT AGG CCG CGGGGT GGT GGG GGC CCC CCC Met Gly Cys Pro Ser Val Ala Arg Pro Arg Gly GlyGly Gly Pro Pro TAGCTAACTAAAGATCC 10P1 (SEQ ID NO:96) Stp Stp Stp Bam/Bg(SEQ ID NO:97)

EXAMPLE 4 Use of NF-kB and NFAT in Signalling

The NFkB/IkB complex is the classic pro-inflammatory second messengersystem, known to be involved as a positive regulator of a number ofpro-inflammatory processes and cytokines. These include, but are notlimited to, IL-1, IL-6, IL-8, and TNF-α. As well, anti-inflammatoryinterleukins, such as IL4, can lead to direct down-modulation of NF-kBin synovial fibroblasts and concomitant downregulation of IL-6production. The NF-kB/IkB complex is a widespread, acute-phase,rapid-response transcriptional activation system. It operates in mostcell types tested, but leads to different outcomes dependent upon thecell type and the nature of the initiating stimulus. Activators of NF-kBinclude LPS, TNF-α, IL-1, inducers of T cell activation, proteinsynthesis inhibitors, phorbol esters, and a-IgM. Other inducers includethe viruses Adenovirus, HTLV I, cytomegalovirus, Sendai, and Herpessimplex I, agents that cause cellular damage such as ultraviolet lightand peroxides, and phosphatase inhibitors such as okadaic acid. Theseinducers act through PKA and PKC-dependent pathways, double-strandRNA-dependent kinase, and other pathways. Pharmacologic regulators ofNF-kB, such as salicylate and glucocorticoids, act by either preventingIkB-α degradation or lead to upregulation of IkB-a transcription andsteady-state levels, thereby acting to prevent the activation of thiscritical factor.

NF-kB (Nuclear Factor that binds to the k locus B site) is present inthe cytoplasm of most cells in an inactive form complexed to IkB(Inhibitor of NF-kB). Certain stimuli received by cells are processed bycellular signaling mechanisms and integrated in the specificphosphorylation of IkB and its degradation. The regulation of IkB-afunction is through a Signal Response Element (SRE) in the aminoterminus of the molecule. Phosphorylation of serine residues 32 and 36leads to recognition of the IkB-a molecule by the ubiquitinationmachinery, release of NF-kB to the nucleus, and degradation of IkB.Therefore, dependent upon the phosphorylation/degradative state of IkB,NF-kB is either maintained in the cytoplasm or released to the nucleus.In the nucleus NF-kB binds to a consensus DNA motif found near theregulatory regions of many characterized genes and therein acts as atranscriptional regulator. Importantly, from the point of view ofinfectious disease, NF-kB is a primary activator of the HumanImmunodeficiency Virus (HIV). Suitable induced genes include TNF-α andIL-6.

Biochemically, NF-kB is defined as a heterodimer of two polypeptides,p50 and p65, of corresponding molecular mass 50 and 65 kD, respectively.p50 is processed from a 105 kD precursor protein by an as yetuncharacterized mechanism. p65 is the receptor for IkB and is themolecule through which IkB exerts its inhibitory/regulatory effects onNF-kB. These are the prototypic transcription factors that define alarge family of classical Rel/NF-kB factors.

Cloning of the p50 and p65 components of NF-kB led to the discovery of afamily of related factors, termed Rel. Both p50 and p65 have a 300 aminomotif (Rel) at their amino termini that was originally described in theproto-oncogene c-rel and the Drosophila axis-determining gene, Dorsal.The family of polypeptides revealed by p50 and p65 have overlappingDNA-binding specificities, differential tissue distribution, and complexregulatory phenomena. p105(p50) is representative of theankyrin-motif-containing Rel proteins that are processed in thecytoplasm to smaller proteins lacking the carboxyl terminus. Thecarboxyl terminus of p105 shows structural and functional homologies toIkB (which also has ankyrin motifs) and functions with an IkB-likeactivity both in cis and in trans. p65 is representative of a secondgroup of Rel proteins that have divergent carboxyl termini--theseregions have been suggested to encode transcriptional activationdomains. The 300 amino acids of Rel domains manifest four importantfunctions: 1) DNA-binding in the roughly amino-terminal 1/3 of thedomain, 2) dimerization in the carboxyl portion of the domain, 3)interaction with ankyrin-containing IkB-like proteins, and 4)nuclear-localizing signal at the carboxyl terminus of the Rel domain. Inp50 the Rel domain also includes a transcriptional activation domain.

NFAT, the Nuclear Factor of Activated T Cells (NFAT), is the immediateearly acute phase response factor for T cell activation. Inhibition ofNFAT by cyclosporin A (CsA) leads to blockade of IL-2 production andloss of T cell commitment to activation.

NFAT, a critical component of pro-inflammatory events carried out by Tcells, is also the factor blocked by CsA in transplantation. Uponcloning NFAT it was clear contains a region of the molecule implicatedin DNA binding that has significant homology to the Rel family ofproteins. Based on structural considerations, homology comparisons, andsimilar modes of action, as well as genomic structures of the moleculesindicate similar intron/exon boundaries in NF-kB and NFAT families, thusindicating that NFAT actually belongs to the Rel family of factors bylineal descent and that its interaction with pro-inflammatorytranscriptional regulators of the bZIP family would follow a general setof rules common to the NF-kB/bZIP interactions.

We have shown that NFAT is involved in pro-inflammatory response tomitogens in activation of HIV-1 (S. Kinoshita and G.P.N, submitted) andthat the binding of NFAT to sites overlapping the NF-kB sites of HIV-1is responsible for this process. This work follows on work by othersshowing that NFAT can regulate TNF-a activation in interaction withATF-2/Jun and GM-CSF. Interestingly, NFAT also appears to be involved inregulation of mast cell release of IL4, an important regulator ofpro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6. The activityof NFAT in these systems has all shown to be pharmacologically modulatedby CsA. Thus, although NFAT was originally discovered as a T cellspecific factor, it was later found to be responsible for a host ofimmediate early, acute phase response activities, as well as directregulation of IL4.

Therefore, the extended Rel families of NF-kB and NFAT make attractivetargets for inhibition and modulation of pro-inflammatory action. Theirinvolvement in numerous regulatory pathways and their decisive roles insuch processes, including the specific interactions they elaborate withbZIP proteins, make them attractive specific targets for inhibition.

Reporter Genes for detection of TNF-α and IL-1 promoter activity.

We designed a retrovirus-based luciferase reporter-gene system driven bya minimal promoter and two Igk NF-kB sites. In the constructs presentedhere, the deletions I introduced were more extensive than thosepreviously published, since preliminary experiments showed that residualenhancer activity resided in commonly available deletion constructs(Nolan, Saksela and Baltimore, unpublished). The vectors designed werepSinII-luc (containing a luciferase gene in the retroviral senseorientation to test for residual promoter activity in the constructbackbone), pSinII-fosluc (identical to pSinII luc except contains aminimal fos promoter element to test for residual enhancer activity inconstruct backbone), and pSinII-2kBfosluc (derived from pSinII-foslucwith 2 Igk kB sites cloned 5' proximal to the fos minimal promoter as areporter for NF-kB activity). These three vectors used to infect 1×10⁶70Z/3 cells. 70Z/3 is a murine pre B cell line originally used in theinitial characterization of NF-kB. After 48 hours, the infected cellswere split into two fractions (stimulated with LPS and unstimulated).Six hours later, cell extracts were prepared and assayed for luciferaseactivity (extract representing ˜10⁴ cells was used for each point). Theresults showed that SinII-luc showed no indiction, SinII-fosluc showedroughly a one-fold increase, and SinII-2kBfosluc showed a four foldinduction in lucerifase activity. Accordingly, retrovirally basedreporter constructs can be used to sensitively report NF-kB activity innative chromatin. It now becomes possible to combine reporter genetechnology with retroviral delivery of effector peptides. Unstimulatedcells and stimulated controls (uninfected and SinII-luc) showed littleor no activity. Importantly, then, retroviral delivery did not result insignificant background induction of NF-kB activity, a problem with othertransfection procedures. The SinII-luc and pSinII-fosluc controls showsno significant residual promoter or enhancer activity in the construct.No significant readthrough from endogenous genomic loci or endogenousenhancer activity that might obscure readings was detected. These latterresults are consistent with previous work using gene search retrovirusesemploying lacZ and flow cytometry. In these studies less than 0.1% ofrandom integration events showed endogenous cis-regulation of theintegrated constructs.

These construct designs will be used as the basis for rapid creation andtesting of TNF-α and IL-1 promoter studies in T cells, macrophages, andsynovial cells. We will incorporate in the place of luciferase eitherthe lacZ or GFP cDNAs for FACS-based assay. We will place up to three tofour kilobases of TNF-α or IL-1 promoter region in place of the minimalpromoter employed here. These constructs will be used as a proxy measureof endogenous TNF-α and IL-1 promoter activity and will serve to allowfor searches for peptides from our libraries that act upon NF-kB or NFATas well as unknown signaling pathways that are independent of NF-kB orNFAT critical to TNF-α and IL-1 signaling.

The B cell lines to be used are 70Z/3. T cells to be used are humanJurkat.

Macrophage lines to be used are Raw 309 and the P388D1 line which ishighly responsive to PMA induction of secreted IL-1. Synovial cells tobe used are HIG-82 and can be activated with IL-1 to inducemetalloproteases and with TNF-α to induce NF-kB. IL-1 induction ofmetalloproteases acts through NF-kB on collagenase and othermetalloproteases of this group. Thus, we have shown that β-gal fused toIkB-α and delivered via a retrovirus to cells responds to stimuli thatdegrade IkB-α as follows: a) 70Z/3 pre-B cells were infected with aretrovirus expressing a fusion of β-gal to either wild-type IkB-α or aninactive, dominant negative IkB-α; infection efficiency wasapproximately 30%. Cells were stimulated with LPS for varying times andthen loaded with FDG for measure of b-gal expression by FACS. b) Cellsfrom (a) were induced for maximal LPS induction of IkB-α degradation andtreated with either salicylate or control. Salicylate blockeddegradation of the β-gal-IkB fusion to the same extent as the dominantnegative IkB-α.

Direct detection in living cells of steady state levels of IkB-α.

At the first approach, NF-kB activation will be measured using our newlydeveloped IkB-α mobile reporter system described above. In thisapproach, the N-terminus of IkB-α has been translationally fused to thelacZ gene. In mammalian cells, β-galactosidase expression can bemeasured using the Fluorescence activated Cell Sorter (FACS) on a cellby cell basis. By coupling β-gal to IkB-α, the stability of β-gal isfunctionally dependent upon IkB-α. Since signals in cells that activateNF-kB lead to the degradation of IkB, β-gal was similarly degraded; asabove, cells were infected with a retrovirus containing a β-gal-IkB-αfusion and induced them with stimuli that lead to activation of NF-kB.We can use the cell sorter to distinguish cells that have degraded IkB-αon a REAL-TIME basis, and not through activation of proxy reportergenes. These lines were shown to respond accordingly after treatmentwith the anti-inflammatory agent salicylate (aspirin) which has beenshown to be a direct inhibitor of NF-kB activation. We have used thisand related protocols in B cells to select for novel mutants of IkB-αand have thereby defined new regions of the IkB-α molecule that respondto differential signaling (J. Caldwell and G. Nolan, unpublished).

1×10⁷ cells carrying the reporter will be infected at high efficiencywith the molecular libraries described herein. Cells will be stimulatedwith LPS, TNF-α, IL-1 or PMA, and then used to select by FACS for thosecells that DO NOT degrade β-gal. After growing out of the cells, thepopulation will be restimulated as before and sorted again. Cells willbe sorted until the population is 100% heritable for the lack ofdegradation phenotype. Inserts will be rescued, recloned into aretrovirus construct, and then screened again until a trans-phenotypecan be confirmed. Peptides will be sequenced as noted.

Selection NFAT-deficiency using cell-death induction by NFAT dependentpathways

We have devised a system for selecting for blockade of NFAT signaling incells that can be employed with our retroviral libraries. The system isbased upon findings by Serafini and colleagues in which they were ableto create a cell line whose death was dependent upon activation of NFAT.Cells stimulated by activators of T cells or NFAT lead to activation ofNFAT and its translocation into the nucleus. Activation leads toinduction of the diphtheria toxin A gene such that the cells undergorapid cell death. This is shown using Propidium iodide as a measure ofcell viability. Thus, in a large population, those cells that areblocked for NFAT activation by peptides that interfere with thesignaling system will survive. Serafini and colleagues used the approachto select for mutants in T cells signaling. We will use this provenNFAT-dipA systemin our peptide selections.

Again, cells will be infected as above with appropriate peptidelibraries and screened for blockade of NFAT signaling. This basicapproach, if successful, might be similarly applied to TNF-α or IL-1signaling.

There is expectation that signaling systems exist whose purpose is toprovide either pro-inflammatory and anti-inflammatory signaling. Asnoted above, IL-4 for instance can blockade IL-6 signaling in cells.Induction of glucocorticoid expression leads to upregulation of IkB andthereby blocks NF-kB activation. Activation of anti-oxidant pathways iswell known to be similarly anti-inflammatory. Salicylate blocks NF-kBthrough regulation of cellular oxygenase levels. Although the peptidesearches outlined above might find players in such pathwaysintracellularly, we desire to search for surface molecules that mightinitiate such protective cascades.

The peptide libraries in constructs for secreted peptides and tetheredpeptides will be used in T cell, macrophage, and B cell systems toselect for blockade OR activation of NF-kB induction. Stimuli willinclude TNF-α and IL-1 for blockade. Activation will utilize theFACS-based systems in "reverse". That is, we will look for peptideswhose expression leads to constitutive activation of and NF-kB reporterconstruct. In this case the reporter construct can be a TNF-a reporterdriving lacZ or GFP. The construct can similarly be IL-1 driving lacZ orGFP. For endogenous loci, we can select for cells that induce VCAM orICAM-1 expression after IL-1 signaling by FACS, both known to bepro-inflammatory responders. Again, both positive AND negative selectioncan be employed. For cells expressing tethered peptides, the selectionis straightforward as the intracellular peptides above. Post-definitionof the peptide sequence, it will be necessary to synthesize the peptidewithout the tether synthetically and determine if the peptide can workin the absence of the tether.

For secreted peptides the setup is more difficult, as the responder cellmust display the phenotype and we must trace the peptide back to theSECRETING cell. For this approach we can use any reporter gene orendogenous gene in the target cells as the readout. The cells to beinfected and which will secrete the peptides will be NIH3T3. 1×10⁷ 3T3cells will be infected with a fully representative library as outlinedabove. Cells post-infection will be allowed to form colonies of up to10-20 cells. At this point media will be removed and the cells will beoverlayed with a thin layer of 0.25% agar in media. Once solidified, athin, porous membrane will be placed over the cells, and we will thenoverlay on this plate the responder cells at high density, also in 0.3%agar. Plates and membranes will be marked with indigo black. In this waysecreted product can diffuse to the responder cells. For selection ofPRO-inflammatory secreted peptides, after 48 hours responder cells willbe lifted from the plate on the membrane and the membrane/cells/agarwill be flipped onto a correspondingly sized nitro-cellulose membrane.Cells will be lysed in situ by Sarcosyl or other appropriate detergentand then applied on the membrane to a high-salt solution and suctionbelow the nitrocellulose. In this way cellular proteins will leach outof the agar matrix and bind to the nitrocellulose. The nitrocellulosecan then be treated like a "Western" for induction or blockade of any ofa number of different cellular proteins. In initial tests we will usereporter genes driving enzymes such as b-gal or alkaline phosphatase toensure assay sensitivity. As we perfect the assay it should be possibleto set up direct measures of certain endogenous loci (such as TNF-α,NF-kB p65, etc.). Once cell areas on the membrane are noted, they can betraced back to the secretion cells by the indigo marking of the platesand alignment. NIH-3T3 cell "patches" corresponding to the appropriatearea can be picked, expanded, and retested. As a positive control,viruses expressing TNF-A or IL-1 will be used in initial scaled mock-upsto calibrate the sensitivity of the search for pro-inflammatorypeptides.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS:  102                                     - - <210> SEQ ID NO 1                                                        <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random             sequence.                                                               <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (7)..(35)                                                     <223> OTHER INFORMATION: The n(s) at positions                                      7,8,10,11,13,14,16,17,19,20,22,23,25,26,28,29,31 - #,3                        2,34,35 can be any nucleic acid.                                         - - <400> SEQUENCE: 1                                                         - - atgggannkn nknnknnknn knnknnknnk nnknnkgggg ggcccccc  - #                    48                                                                         - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 16                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                               <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (3)..(12)                                                     <223> OTHER INFORMATION: The Xaa(s) at positions - #3-12 can be any          amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 2                                                         - - Met Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Gly Gly Pro        Pro                                                                               1               5 - #                 10 - #                 15             - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: molecular           flexibility/stability sequence.                                          - - <400> SEQUENCE: 3                                                         - - Gly Gly Pro Pro                                                            1                                                                            - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 61                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: coiled-co    il                                                                                   structure.                                                               - - <400> SEQUENCE: 4                                                         - - Met Gly Cys Ala Ala Leu Glu Ser Glu Val Se - #r Ala Leu Glu Ser Glu        1               5 - #                 10 - #                 15              - - Val Ala Ser Leu Glu Ser Glu Val Ala Ala Le - #u Gly Arg Gly Asp Met                   20     - #             25     - #             30                  - - Pro Leu Ala Ala Val Lys Ser Lys Leu Ser Al - #a Val Lys Ser Lys Leu               35         - #         40         - #         45                      - - Ala Ser Val Lys Ser Lys Leu Ala Ala Cys Gl - #y Pro Pro                       50             - #     55             - #     60                          - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: loop                structure.                                                               - - <400> SEQUENCE: 5                                                         - - Gly Arg Gly Asp Met Pro                                                    1               5                                                            - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 69                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: minibody            presentation structure.                                                  - - <400> SEQUENCE: 6                                                         - - Met Gly Arg Asn Ser Gln Ala Thr Ser Gly Ph - #e Thr Phe Ser His Phe        1               5 - #                 10 - #                 15              - - Tyr Met Glu Trp Val Arg Gly Gly Glu Tyr Il - #e Ala Ala Ser Arg His                   20     - #             25     - #             30                  - - Lys His Asn Lys Tyr Thr Thr Glu Tyr Ser Al - #a Ser Val Lys Gly Arg               35         - #         40         - #         45                      - - Tyr Ile Val Ser Arg Asp Thr Ser Gln Ser Il - #e Leu Tyr Leu Gln Lys           50             - #     55             - #     60                          - - Lys Lys Gly Pro Pro                                                       65                                                                            - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: nuclear             localization sequence.                                                   - - <400> SEQUENCE: 7                                                         - - Pro Lys Lys Lys Arg Lys Val                                                1               5                                                            - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: nuclear             loclization sequence.                                                    - - <400> SEQUENCE: 8                                                         - - Ala Arg Arg Arg Arg Pro                                                    1               5                                                            - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: nuclear             localization sequence.                                                   - - <400> SEQUENCE: 9                                                         - - Glu Glu Val Gln Arg Lys Arg Gln Lys Leu                                    1               5 - #                 10                                     - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: nuclear             localization sequence.                                                   - - <400> SEQUENCE: 10                                                        - - Glu Glu Lys Arg Lys Arg Thr Tyr Glu                                        1               5                                                            - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: nuclear             localization sequence.                                                   - - <400> SEQUENCE: 11                                                        - - Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Al - #a Gly Gln Ala Lys Lys        1               5 - #                 10 - #                 15              - - Lys Lys Leu Asp                                                                       20                                                                - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 31                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: signal              sequence.                                                                - - <400> SEQUENCE: 12                                                        - - Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Le - #u Asn Leu Leu Leu Leu        1               5 - #                 10 - #                 15              - - Gly Glu Ser Ile Leu Gly Ser Gly Glu Ala Ly - #s Pro Gln Ala Pro                       20     - #             25     - #             30                  - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: signal              sequence.                                                                - - <400> SEQUENCE: 13                                                        - - Met Ser Ser Phe Gly Tyr Arg Thr Leu Thr Va - #l Ala Leu Phe Thr Leu        1               5 - #                 10 - #                 15              - - Ile Cys Cys Pro Gly                                                                   20                                                                - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 51                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     transmembrane domain sequence.                                           - - <400> SEQUENCE: 14                                                        - - Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gl - #y Ser Val Lys Gly Thr        1               5 - #                 10 - #                 15              - - Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Tr - #p Ala Pro Leu Ala Gly                   20     - #             25     - #             30                  - - Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Il - #e Thr Leu Ile Cys Tyr               35         - #         40         - #         45                      - - His Ser Arg                                                                   50                                                                        - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 33                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     transmembrane sequence.                                                  - - <400> SEQUENCE: 15                                                        - - Met Val Ile Ile Val Thr Val Val Ser Val Le - #u Leu Ser Leu Phe Val        1               5 - #                 10 - #                 15              - - Thr Ser Val Leu Leu Cys Phe Ile Phe Gly Gl - #n His Leu Arg Gln Gln                   20     - #             25     - #             30                  - - Arg                                                                       - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 37                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: membrane            anchor sequence.                                                         - - <400> SEQUENCE: 16                                                        - - Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Th - #r Thr Arg Leu Leu Ser        1               5 - #                 10 - #                 15              - - Gly His Thr Cys Phe Thr Leu Thr Gly Leu Le - #u Gly Thr Leu Val Thr                   20     - #             25     - #             30                  - - Met Gly Leu Leu Thr                                                               35                                                                    - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     myristylation sequence.                                                  - - <400> SEQUENCE: 17                                                        - - Met Gly Ser Ser Lys Ser Lys Pro Lys Asp Pr - #o Ser Gln Arg                1               5 - #                 10                                     - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 26                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     palmitoylation sequence.                                                 - - <400> SEQUENCE: 18                                                        - - Leu Leu Gln Arg Leu Phe Ser Arg Gln Asp Cy - #s Cys Gly Asn Cys Ser        1               5 - #                 10 - #                 15              - - Asp Ser Glu Glu Glu Leu Pro Thr Arg Leu                                               20     - #             25                                         - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     palmitoylation sequence.                                                 - - <400> SEQUENCE: 19                                                        - - Lys Gln Phe Arg Asn Cys Met Leu Thr Ser Le - #u Cys Cys Gly Lys Asn        1               5 - #                 10 - #                 15              - - Pro Leu Gly Asp                                                                       20                                                                - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 19                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     palmitoylation sequence.                                                 - - <400> SEQUENCE: 20                                                        - - Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly Cy - #s Met Ser Cys Lys Cys        1               5 - #                 10 - #                 15              - - Val Leu Ser                                                               - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     lysosomal degradation sequence.                                          - - <400> SEQUENCE: 21                                                        - - Lys Phe Glu Arg Gln                                                        1               5                                                            - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 36                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: lysosomal           membrane sequence.                                                       - - <400> SEQUENCE: 22                                                        - - Met Leu Ile Pro Ile Ala Gly Phe Phe Ala Le - #u Ala Gly Leu Val Leu        1               5 - #                 10 - #                 15              - - Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Ly - #s Arg Ser His Ala Gly                   20     - #             25     - #             30                  - - Tyr Gln Thr Ile                                                                   35                                                                    - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 35                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: lysosomal           degradation sequence.                                                    - - <400> SEQUENCE: 23                                                        - - Leu Val Pro Ile Ala Val Gly Ala Ala Leu Al - #a Gly Val Leu Ile Leu        1               5 - #                 10 - #                 15              - - Val Leu Leu Ala Tyr Phe Ile Gly Leu Lys Hi - #s His His Ala Gly Tyr                   20     - #             25     - #             30                  - - Glu Gln Phe                                                                       35                                                                    - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     mitochondrial matrix sequence.                                           - - <400> SEQUENCE: 24                                                        - - Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Ar - #g Val Gln Pro Ser Leu        1               5 - #                 10 - #                 15              - - Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Th - #r                                       20     - #             25                                         - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 25                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     mitochondrial inner membrane sequence.                                   - - <400> SEQUENCE: 25                                                        - - Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Ph - #e Lys Pro Ala Thr Arg        1               5 - #                 10 - #                 15              - - Thr Leu Cys Ser Ser Arg Tyr Leu Leu                                                   20     - #             25                                         - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 64                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     mitochondrial intermembrane sequence.                                    - - <400> SEQUENCE: 26                                                        - - Met Phe Ser Met Leu Ser Lys Arg Trp Ala Gl - #n Arg Thr Leu Ser Lys        1               5 - #                 10 - #                 15              - - Ser Phe Tyr Ser Thr Ala Thr Gly Ala Ala Se - #r Lys Ser Gly Lys Leu                   20     - #             25     - #             30                  - - Thr Gln Lys Leu Val Thr Ala Gly Val Ala Al - #a Ala Gly Ile Thr Ala               35         - #         40         - #         45                      - - Ser Thr Leu Leu Tyr Ala Asp Ser Leu Thr Al - #a Glu Ala Met Thr Ala           50             - #     55             - #     60                          - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 41                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     mitochondrial outer membrane sequence.                                   - - <400> SEQUENCE: 27                                                        - - Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Al - #a Ile Leu Ala Thr Val        1               5 - #                 10 - #                 15              - - Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Ty - #r Tyr Tyr Asn Gln Leu                   20     - #             25     - #             30                  - - Gln Gln Gln Gln Gln Arg Gly Lys Lys                                               35         - #         40                                             - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: endoplasm    ic                                                                                   reticulum sequence.                                                      - - <400> SEQUENCE: 28                                                        - - Lys Asp Glu Leu                                                            1                                                                            - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: endoplasm    ic                                                                                   reticulum sequence.                                                      - - <400> SEQUENCE: 29                                                        - - Leu Tyr Leu Ser Arg Arg Ser Phe Ile Asp Gl - #u Lys Lys Met Pro            1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 19                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     farnesylation sequence.                                                  - - <400> SEQUENCE: 30                                                        - - Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly Cy - #s Met Ser Cys Lys Cys        1               5 - #                 10 - #                 15              - - Val Leu Ser                                                               - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     geranylgeranylation sequence.                                            - - <400> SEQUENCE: 31                                                        - - Leu Thr Glu Pro Thr Gln Pro Thr Arg Asn Gl - #n Cys Cys Ser Asn            1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     destruction sequence.                                                    - - <400> SEQUENCE: 32                                                        - - Arg Thr Ala Leu Gly Asp Ile Gly Asn                                        1               5                                                            - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:secretory            sequence.                                                                - - <400> SEQUENCE: 33                                                        - - Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Al - #a Leu Ser Leu Ala Leu        1               5 - #                 10 - #                 15              - - Val Thr Asn Ser                                                                       20                                                                - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: secretory           sequence.                                                                - - <400> SEQUENCE: 34                                                        - - Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Le - #u Ala Phe Gly Leu Leu        1               5 - #                 10 - #                 15              - - Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ph - #e Pro Thr                               20     - #             25                                         - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: secretory           sequence.                                                                - - <400> SEQUENCE: 35                                                        - - Met Ala Leu Trp Met Arg Leu Leu Pro Leu Le - #u Ala Leu Leu Ala Leu        1               5 - #                 10 - #                 15              - - Trp Gly Pro Asp Pro Ala Ala Ala Phe Val As - #n                                       20     - #             25                                         - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 18                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: secretory           sequence.                                                                - - <400> SEQUENCE: 36                                                        - - Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Al - #a Phe Val Ala Gly Asp        1               5 - #                 10 - #                 15              - - Gln Ile                                                                   - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:secretory            sequence.                                                                - - <400> SEQUENCE: 37                                                        - - Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Le - #u Phe Phe Leu Leu Ala        1               5 - #                 10 - #                 15              - - Cys Ala Gly Asn Phe Val His Gly                                                       20                                                                - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: stability           sequence.                                                               <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (3)..(6)                                                      <223> OTHER INFORMATION: The Xaa(s) at positions - #3-6 can be any                  amino acid.                                                              - - <400> SEQUENCE: 38                                                        - - Met Gly Xaa Xaa Xaa Xaa Gly Gly Pro Pro                                    1               5 - #                 10                                     - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: linker              sequence.                                                                - - <400> SEQUENCE: 39                                                        - - Gly Ser Gly Gly Ser                                                        1               5                                                            - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: linker              sequence.                                                                - - <400> SEQUENCE: 40                                                        - - Gly Gly Gly Ser                                                            1                                                                            - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 124                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (115)..(120)                                                  <223> OTHER INFORMATION: The Xaa(s) at postions - #115-120 can be any        amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 41                                                        - - Met Arg Pro Leu Ala Gly Gly Glu His Thr Me - #t Ala Ser Pro Leu        Thr                                                                               1               5 - #                 10 - #                 15             - - Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu Gl - #y Glu Ser Ile Ile Leu                   20     - #             25     - #             30                  - - Gly Ser Gly Pro Gln Arg Pro Glu Asp Cys Ar - #g Pro Arg Gly Ser Val               35         - #         40         - #         45                      - - Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Il - #e Tyr Ile Trp Ala Pro           50             - #     55             - #     60                          - - Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Se - #r Leu Ile Ile Thr Leu       65                 - # 70                 - # 75                 - # 80       - - Ile Cys Tyr His Ser Arg Gly Ser Gly Gly Se - #r Gly Ser Gly Gly Ser                       85 - #                 90 - #                 95              - - Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gl - #y Ser Gly Gly Ser Gly                  100      - #           105      - #           110                  - - Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Gly Gly Pr - #o Pro                              115          - #       120                                             - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 173                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (140)..(145)                                                  <223> OTHER INFORMATION: The Xaa(s) at positions - #140-145 can be any              amino acid.                                                              - - <400> SEQUENCE: 42                                                        - - Met Arg Pro Leu Ala Gly Gly Glu His Thr Me - #t Ala Ser Pro Leu Thr        1               5 - #                 10 - #                 15              - - Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu Gl - #y Glu Ser Ile Ile Leu                   20     - #             25     - #             30                  - - Gly Ser Gly Pro Gln Arg Pro Glu Asp Cys Ar - #g Pro Arg Gly Ser Val               35         - #         40         - #         45                      - - Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Il - #e Tyr Ile Trp Ala Pro           50             - #     55             - #     60                          - - Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Se - #r Leu Ile Ile Thr Leu       65                 - # 70                 - # 75                 - # 80       - - Ile Cys Tyr His Ser Arg Gly Ser Gly Gly Se - #r Gly Ser Gly Gly Ser                       85 - #                 90 - #                 95              - - Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gl - #y Ser Gly Gly Ser Gly                  100      - #           105      - #           110                  - - Gly Gly Cys Ala Ala Leu Glu Ser Glu Val Se - #r Ala Leu Glu Ser Glu              115          - #       120          - #       125                      - - Val Ala Ser Leu Glu Ser Glu Val Ala Ala Le - #u Xaa Xaa Xaa Xaa Xaa          130              - #   135              - #   140                          - - Xaa Leu Ala Ala Val Lys Ser Lys Leu Ser Al - #a Val Lys Ser Lys Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Ser Val Lys Ser Lys Leu Ala Ala Cys Gl - #y Pro Pro                                  165  - #               170                                     - -  - - <210> SEQ ID NO 43                                                  <211> LENGTH: 127                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic.                                                                      <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (38)..(43)                                                    <223> OTHER INFORMATION: The Xaa(s) at positions - #38-43 can be any        amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 43                                                        - - Met Arg Pro Leu Ala Gly Gly Glu His Thr Me - #t Ala Ser Pro Leu        Thr                                                                               1               5 - #                 10 - #                 15             - - Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu Gl - #y Glu Ser Ile Ile Leu                   20     - #             25     - #             30                  - - Gly Ser Gly Gly Gly Xaa Xaa Xaa Xaa Xaa Xa - #a Gly Gly Ser Gly Gly               35         - #         40         - #         45                      - - Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Se - #r Gly Ser Gly Gly Ser           50             - #     55             - #     60                          - - Gly Ser Gly Gly Ser Gly Gly Gly Pro Gln Ar - #g Pro Glu Asp Cys Arg       65                 - # 70                 - # 75                 - # 80       - - Pro Arg Gly Ser Val Lys Gly Thr Gly Leu As - #p Phe Ala Cys Asp Ile                       85 - #                 90 - #                 95              - - Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Va - #l Ala Leu Leu Leu Ser                  100      - #           105      - #           110                  - - Leu Ile Ile Thr Leu Ile Cys Tyr His Ser Ar - #g Gly Gly Pro Pro                  115          - #       120          - #       125                      - -  - - <210> SEQ ID NO 44                                                  <211> LENGTH: 177                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (63)..(68)                                                    <223> OTHER INFORMATION: The Xaa(s) at positions - #63-68 can be any         amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 44                                                        - - Met Arg Pro Leu Ala Gly Gly Glu His Thr Me - #t Ala Ser Pro Leu        Thr                                                                               1               5 - #                 10 - #                 15             - - Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu Gl - #y Glu Ser Ile Ile Leu                   20     - #             25     - #             30                  - - Gly Ser Gly Gly Gly Cys Ala Ala Leu Glu Se - #r Glu Val Ser Ala Leu               35         - #         40         - #         45                      - - Glu Ser Glu Val Ala Ser Leu Glu Ser Glu Va - #l Ala Ala Leu Xaa Xaa           50             - #     55             - #     60                          - - Xaa Xaa Xaa Xaa Leu Ala Ala Val Lys Ser Ly - #s Leu Ser Ala Val Lys       65                 - # 70                 - # 75                 - # 80       - - Ser Lys Leu Ala Ser Val Lys Ser Lys Leu Al - #a Ala Cys Gly Gly Ser                       85 - #                 90 - #                 95              - - Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser Gl - #y Gly Ser Gly Ser Gly                  100      - #           105      - #           110                  - - Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Pr - #o Gln Arg Pro Glu Asp              115          - #       120          - #       125                      - - Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gl - #y Leu Asp Phe Ala Cys          130              - #   135              - #   140                          - - Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Il - #e Cys Val Ala Leu Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr Hi - #s Ser Arg Gly Gly        Pro                                                                                             165  - #               170  - #               175             - - Pro                                                                       - -  - - <210> SEQ ID NO 45                                                  <211> LENGTH: 47                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (38)..(43)                                                    <223> OTHER INFORMATION: The Xaa(s) at positions - #38-43 can be any         amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 45                                                        - - Met Arg Pro Leu Ala Gly Gly Glu His Arg Me - #t Ala Ser Pro Leu        Thr                                                                               1               5 - #                 10 - #                 15             - - Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu Gl - #y Glu Ser Ile Ile Leu                   20     - #             25     - #             30                  - - Gly Ser Gly Gly Gly Xaa Xaa Xaa Xaa Xaa Xa - #a Gly Gly Pro Pro                   35         - #         40         - #         45                      - -  - - <210> SEQ ID NO 46                                                  <211> LENGTH: 95                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (62)..(67)                                                    <223> OTHER INFORMATION: The Xaa(s) at positions - #62-67 can be any         amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 46                                                        - - Met Arg Pro Leu Ala Gly Gly Glu His Thr Me - #t Ala Ser Pro Leu        Thr                                                                               1               5 - #                 10 - #                 15             - - Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu Gl - #y Glu Ser Ile Ile Leu                   20     - #             25     - #             30                  - - Gly Ser Gly Gly Gly Ala Ala Leu Glu Ser Gl - #u Val Ser Ala Leu Glu               35         - #         40         - #         45                      - - Ser Glu Val Ala Ser Leu Glu Ser Glu Val Al - #a Ala Leu Xaa Xaa Xaa           50             - #     55             - #     60                          - - Xaa Xaa Xaa Leu Ala Ala Val Lys Ser Lys Le - #u Ser Ala Val Lys Ser       65                 - # 70                 - # 75                 - # 80       - - Lys Leu Ala Ser Val Lys Ser Lys Leu Ala Al - #a Cys Gly Pro Pro                           85 - #                 90 - #                 95              - -  - - <210> SEQ ID NO 47                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)..(9)                                                      <223> OTHER INFORMATION: The Xaa(s) at positions - #1-3, 6, 8, 9 can be             any amino acid.                                                          - - <400> SEQUENCE: 47                                                        - - Xaa Xaa Xaa Pro Pro Xaa Pro Xaa Xaa                                        1               5                                                            - -  - - <210> SEQ ID NO 48                                                  <211> LENGTH: 63                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (7)..(20)                                                     <223> OTHER INFORMATION: The n(s) at positions                                      7,8,10,11,13,14,16,17,19,20 can be any - #nucleic acid.                  - - <400> SEQUENCE: 48                                                        - - atgggcnnkn nknnknnknn kagacctctg cctccasbkg ggsbksbkgg ag -             #gcccacct     60                                                                 - - taa                  - #                  - #                  - #                 63                                                                  - -  - - <210> SEQ ID NO 49                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (3)..(16)                                                     <223> OTHER INFORMATION: The Xaa(s) at postions - #3-7, 13,15,16 can be      any                                                                                   amino acid.                                                              - - <400> SEQUENCE: 49                                                        - - Met Gly Xaa Xaa Xaa Xaa Xaa Arg Pro Leu Pr - #o Pro Xaa Pro Xaa        Xaa                                                                               1               5 - #                 10 - #                 15             - - Gly Gly Pro Pro                                                                       20                                                                - -  - - <210> SEQ ID NO 50                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                               <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (2)..(11)                                                     <223> OTHER INFORMATION: The Xaa(s) at postions - #2-11 can be any amino            acid.                                                                    - - <400> SEQUENCE: 50                                                        - - Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Cys                        1               5 - #                 10                                     - -  - - <210> SEQ ID NO 51                                                  <211> LENGTH: 17                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: epitope      tag                                                                                   sequence.                                                                - - <400> SEQUENCE: 51                                                        - - Met Gly Gly Gly Tyr Pro Tyr Asp Val Pro As - #p Tyr Ala Gly Ser        Leu                                                                               1               5 - #                 10 - #                 15             - - Glx                                                                       - -  - - <210> SEQ ID NO 52                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: PKCa                translocation inhibitor sequence.                                        - - <400> SEQUENCE: 52                                                        - - Gly Lys Gln Lys Thr Lys Thr Ile Lys Gly Pr - #o Pro                        1               5 - #                 10                                     - -  - - <210> SEQ ID NO 53                                                  <211> LENGTH: 92                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                               <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (28)..(56)                                                    <223> OTHER INFORMATION: The n(s) at postions                                       28,29,31,32,34,35,37,38,40,41,43,44,46,47,49,50, - #52                        ,53,55,56 can be any nucleic acid - #.                                   - - <400> SEQUENCE: 53                                                        - - gcttagcaag atctctacgg tggaccknnk nnknnknnkn nknnknnknn kn -             #nknncccc     60                                                                 - - actcccatgg tcctacgtac caccacactg gg       - #                  - #              92                                                                     - -  - - <210> SEQ ID NO 54                                                  <211> LENGTH: 34                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 54                                                        - - gcttagcaag atctgtgtgt cagttagggt gtgg       - #                  -      #        34                                                                      - -  - - <210> SEQ ID NO 55                                                  <211> LENGTH: 47                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random             sequence.                                                               <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)..(24)                                                    <223> OTHER INFORMATION: The n(s) at positions - #23-24 can be any           nucleic                                                                               acid.                                                                    - - <400> SEQUENCE: 55                                                        - - ctggagaacc aggaccatgg gcnnkgggcc cccttaaacc attaaat   - #                    47                                                                        - -  - - <210> SEQ ID NO 56                                                  <211> LENGTH: 71                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                               <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)..(48)                                                    <223> OTHER INFORMATION: The n(s) at positions                                      23,24,26,27,29,30,38,39,44,45,47,48 can be - # any                            nucleic acid.                                                            - - <400> SEQUENCE: 56                                                        - - ctggagaacc aggaccatgg gcnnknnknn kcctcccnnk cctnnknnkg gg -             #ccccctta     60                                                                 - - aaccattaaa t               - #                  - #                      - #       71                                                                  - -  - - <210> SEQ ID NO 57                                                  <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 57                                                        - - tcatgcatcc aatttaatgg tttaag          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 58                                                  <211> LENGTH: 4950                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: retrovira    l                                                                                    vector with presentation construct s - #equence.                         - - <400> SEQUENCE: 58                                                        - - tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tg -             #caaggcat     60                                                                 - - ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag ag -            #agacagca    120                                                                 - - gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc ag -            #ggccaaga    180                                                                 - - acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tc -            #agatgttt    240                                                                 - - ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa cc -            #aatcagtt    300                                                                 - - cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gc -            #ccacaacc    360                                                                 - - cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg ta -            #ttcccaat    420                                                                 - - aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gg -            #gtctcctc    480                                                                 - - agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg ag -            #atttggag    540                                                                 - - acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cg -            #gtcgtttc    600                                                                 - - ctgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cc -            #tgcgtctg    660                                                                 - - tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cg -            #agttctga    720                                                                 - - acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt tt -            #gtggcccg    780                                                                 - - acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tc -            #tggtagga    840                                                                 - - gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt tt -            #ggaaccga    900                                                                 - - agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttctct ct -            #gtctgact    960                                                                 - - gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aa -            #gtttgacc   1020                                                                 - - ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tg -            #tcaagaag   1080                                                                 - - agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg at -            #ggccgcga   1140                                                                 - - gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt tt -            #cacctggc   1200                                                                 - - ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt gg -            #cttttgac   1260                                                                 - - ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tc -            #ctccatcc   1320                                                                 - - gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cc -            #tttatcca   1380                                                                 - - gccctcactc cttctctagg cgccggaatt ccaggaccat gggcgggccc cc -            #ttaaacca   1440                                                                 - - ttaaattggt aaaataaagg atccgtcgac ctgcagccaa gcttatcgat aa -            #aataaaag   1500                                                                 - - attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag gt -            #ttggcaag   1560                                                                 - - ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga at -            #agagaagt   1620                                                                 - - tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga ta -            #tctgtggt   1680                                                                 - - aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc gg -            #tcccgccc   1740                                                                 - - tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tg -            #aaaatgac   1800                                                                 - - cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cg -            #cgcttctg   1860                                                                 - - ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt cc -            #tccgatag   1920                                                                 - - actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tc -            #cgacttgt   1980                                                                 - - ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc ag -            #cgggggtc   2040                                                                 - - tttcattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gc -            #tcacaatt   2100                                                                 - - ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta at -            #gagtgagc   2160                                                                 - - taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cc -            #tgtcgtgc   2220                                                                 - - cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tg -            #ggcgctct   2280                                                                 - - tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg ag -            #cggtatca   2340                                                                 - - gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc ag -            #gaaagaac   2400                                                                 - - atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gc -            #tggcgttt   2460                                                                 - - ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tc -            #agaggtgg   2520                                                                 - - cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cc -            #tcgtgcgc   2580                                                                 - - tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc tt -            #cgggaagc   2640                                                                 - - gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cg -            #ttcgctcc   2700                                                                 - - aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt at -            #ccggtaac   2760                                                                 - - tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc ag -            #ccactggt   2820                                                                 - - aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gt -            #ggtggcct   2880                                                                 - - aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gc -            #cagttacc   2940                                                                 - - ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg ta -            #gcggtggt   3000                                                                 - - ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga ag -            #atcctttg   3060                                                                 - - atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg ga -            #ttttggtc   3120                                                                 - - atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aa -            #gttttaaa   3180                                                                 - - tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aa -            #tcagtgag   3240                                                                 - - gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact cc -            #ccgtcgtg   3300                                                                 - - tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat ga -            #taccgcga   3360                                                                 - - gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aa -            #gggccgag   3420                                                                 - - cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg tt -            #gccgggaa   3480                                                                 - - gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tg -            #ctacaggc   3540                                                                 - - atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc cc -            #aacgatca   3600                                                                 - - aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cg -            #gtcctccg   3660                                                                 - - atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc ag -            #cactgcat   3720                                                                 - - aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gt -            #actcaacc   3780                                                                 - - aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gt -            #caatacgg   3840                                                                 - - gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa ac -            #gttcttcg   3900                                                                 - - gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta ac -            #ccactcgt   3960                                                                 - - gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg ag -            #caaaaaca   4020                                                                 - - ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aa -            #tactcata   4080                                                                 - - ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat ga -            #gcggatac   4140                                                                 - - atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tc -            #cccgaaaa   4200                                                                 - - gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aa -            #ataggcgt   4260                                                                 - - atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ct -            #gacacatg   4320                                                                 - - cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag ac -            #aagcccgt   4380                                                                 - - cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc gg -            #catcagag   4440                                                                 - - cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cg -            #taaggaga   4500                                                                 - - aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga ag -            #ggcgatcg   4560                                                                 - - gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aa -            #ggcgatta   4620                                                                 - - agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc ca -            #gtgccacg   4680                                                                 - - ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga gg -            #ccgttgag   4740                                                                 - - caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc cc -            #ccggccac   4800                                                                 - - ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt gg -            #cgagcccg   4860                                                                 - - atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tg -            #gcgccggt   4920                                                                 - - gatgccggcc acgatgcgtc cggcgtagag         - #                  - #             4950                                                                     - -  - - <210> SEQ ID NO 59                                                  <211> LENGTH: 74                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 59                                                        - - ctggagaacc aggaccatgg gcaagagaaa gggcgatgag gtggatggag tg -             #gggccccc     60                                                                 - - ttaaaccatt aaat              - #                  - #                      - #     74                                                                  - -  - - <210> SEQ ID NO 60                                                  <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     anti-apoptosis sequence.                                                 - - <400> SEQUENCE: 60                                                        - - Met Gly Lys Arg Lys Gly Asp Glu Val Asp Gl - #y Val Gly Pro Pro            1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 61                                                  <211> LENGTH: 74                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                               <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (35)..(48)                                                    <223> OTHER INFORMATION: The n(s) at positions - #35,36,38,39,41,42,47,48           can be any nucleic acid.                                                 - - <400> SEQUENCE: 61                                                        - - ctggagaacc aggaccatgg gcaagagaaa gggcnnknnk nnkgaknnkg tg -             #gggccccc     60                                                                 - - ttaaaccatt aaat              - #                  - #                      - #     74                                                                  - -  - - <210> SEQ ID NO 62                                                  <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                               <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (7)..(11)                                                     <223> OTHER INFORMATION: The Xaa(s) at postions - #7-9,11 can be any         amino                                                                                 acid.                                                                   <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (10)                                                          <223> OTHER INFORMATION: The amino acid at posi - #tion 10 can be           Aspartic                                                                              acid or Glutamic acid.                                                   - - <400> SEQUENCE: 62                                                        - - Met Gly Lys Arg Lys Gly Xaa Xaa Xaa Asp Xa - #a Val Gly Pro Pro           1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 63                                                  <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 63                                                        - - tcatgcatcc aatttaatgg tttaag          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 64                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 64                                                        - - gatcctccct ttatccag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 65                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 65                                                        - - ctacaggtgg ggtctttc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 66                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 66                                                        - - atgggcaaga gaaagggcac ggcgtctgat gctgtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 67                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 67                                                        - - Thr Ala Ser Asp Ala                                                        1               5                                                            - -  - - <210> SEQ ID NO 68                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 68                                                        - - atgggcaaga gaaagggcta tccttctgat gtggtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 69                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 69                                                        - - Tyr Pro Ser Asp Val                                                        1               5                                                            - -  - - <210> SEQ ID NO 70                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 70                                                        - - atgggcaaga gaaagggcac gccttcggat atggtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 71                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 71                                                        - - Thr Pro Ser Asp Met                                                        1               5                                                            - -  - - <210> SEQ ID NO 72                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 72                                                        - - atgggcaaga gaaagggcac ggcttctgat cttgtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 73                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 73                                                        - - Thr Ala Ser Asp Leu                                                        1               5                                                            - -  - - <210> SEQ ID NO 74                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 74                                                        - - atgggcaaga gaaagggctc tgatagggat attgtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 75                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 75                                                        - - Ser Asp Arg Asp Ile                                                        1               5                                                            - -  - - <210> SEQ ID NO 76                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 76                                                        - - atgggcaaga gaaagggctg gttgctagag tttgtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 77                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 77                                                        - - Trp Leu Leu Glu Phe                                                        1               5                                                            - -  - - <210> SEQ ID NO 78                                                  <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 78                                                        - - atgggcaaga gaaagggctg gttgatagag tttgtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 79                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 79                                                        - - Trp Leu Ile Glu Phe                                                        1               5                                                            - -  - - <210> SEQ ID NO 80                                                  <211> LENGTH: 6                                                               <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic     <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)..(6)                                                      <223> OTHER INFORMATION: The n(s) at positions - #1-6 can be any nucleic            acid.                                                                    - - <400> SEQUENCE: 80                                                        - - nnnnnn                 - #                  - #                  -      #            6                                                                   - -  - - <210> SEQ ID NO 81                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 81                                                        - - Ser Tyr Gln Asp Leu                                                        1               5                                                            - -  - - <210> SEQ ID NO 82                                                  <211> LENGTH: 16                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                       <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (3)..(12)                                                     <223> OTHER INFORMATION: The Xaa(s) at positions - #3-12 can be any         amino                                                                                 acid.                                                                    - - <400> SEQUENCE: 82                                                        - - Met Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Gly Gly Pro        Pro                                                                               1               5 - #                 10 - #                 15             - -  - - <210> SEQ ID NO 83                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 83                                                        - - ctgacacaca ttccacag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 84                                                  <211> LENGTH: 122                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 84                                                        - - ggatccagtg tggtggtacg taggaatacc atgggatgtc cgtctgttgc ta -             #ggccgcgg     60                                                                 - - ggtggtgggg gcccccccta gctaactaaa gatcccagtg tggtggtacg ta -            #ggaattcg    120                                                                 - - cc                  - #                  - #                  - #                 122                                                                  - -  - - <210> SEQ ID NO 85                                                  <211> LENGTH: 16                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 85                                                        - - Met Gly Cys Pro Ser Val Ala Arg Pro Arg Gl - #y Gly Gly Gly Pro Pro        1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 86                                                  <211> LENGTH: 112                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 86                                                        - - ggatcccagt gtggtggtac gtaggaatac catgggattg tcttttgtta tt -             #ygtctgca     60                                                                 - - gcatcgtggg ggccccccct agctaactaa agatcccagt gtggtggtac gt - #                112                                                                       - -  - - <210> SEQ ID NO 87                                                  <211> LENGTH: 16                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 87                                                        - - Met Gly Leu Ser Phe Val Ile Leu Ser Ala Al - #a Ser Trp Gly Pro Pro        1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 88                                                  <211> LENGTH: 96                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 88                                                        - - ggatcccagt gtggtggtac gtaggagtac catgggacct ccgatttggt at -             #actcattg     60                                                                 - - gagtcatggg ggccccccct agctaactaa agatcc      - #                       - #       96                                                                     - -  - - <210> SEQ ID NO 89                                                  <211> LENGTH: 16                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 89                                                        - - Met Gly Pro Pro Ile Trp Tyr Thr His Trp Se - #r His Gly Gly Pro        Pro                                                                               1               5 - #                 10 - #                 15             - -  - - <210> SEQ ID NO 90                                                  <211> LENGTH: 95                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 90                                                        - - ggatcccagt gtggtggtac gtaggagtac catggaagtc aggcgtttgt ga -             #atactcgg     60                                                                 - - cataaggggg gcccccccta gctaactaaa gatcc       - #                       - #       95                                                                     - -  - - <210> SEQ ID NO 91                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 91                                                        - - Met Glu Val Arg Arg Leu                                                    1               5                                                            - -  - - <210> SEQ ID NO 92                                                  <211> LENGTH: 126                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 92                                                        - - ccggccgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg ta -            #tgggatct     60                                                                 - - gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aa -            #acgtctag    120                                                                 - - gccccc                 - #                  - #                  -     #          126                                                                  - -  - - <210> SEQ ID NO 93                                                  <211> LENGTH: 107                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 93                                                        - - ggatcccagt gtggtggtac gtaggaatac catgggactt tagccgggcc cc -             #ccctagct     60                                                                 - - aactaaagat cccagtgtgg tggtacgtag gaattcgcca gcacagt   - #                   107                                                                        - -  - - <210> SEQ ID NO 94                                                  <211> LENGTH: 95                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 94                                                        - - ggatcccagt gtggtggtac gtaggaatac atgggaactg ttatggcgat gt -             #cggattag     60                                                                 - - gtcgaggggg gcccccccta gctaactaaa gatcc       - #                       - #       95                                                                     - -  - - <210> SEQ ID NO 95                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 95                                                        - - Met Gly Thr Val Met Ala Met Ser Asp                                        1               5                                                            - -  - - <210> SEQ ID NO 96                                                  <211> LENGTH: 95                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 96                                                        - - ggatccagtg tggtggtacg taggaatacc atgggatgtc cgtctgttgc ta -            #ggccgcgg     60                                                                 - - ggtggtgggg gcccccccta gctaactaaa gatcc       - #                       - #       95                                                                     - -  - - <210> SEQ ID NO 97                                                  <211> LENGTH: 16                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             synthetic                                                                        - - <400> SEQUENCE: 97                                                        - - Met Gly Cys Pro Ser Val Ala Arg Pro Arg Gl - #y Gly Gly Gly Pro        Pro                                                                               1               5 - #                 10 - #                 15             - -  - - <210> SEQ ID NO 98                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)..(5)                                                      <223> OTHER INFORMATION: The Xaa(s) at postions - #1-5 can be any                   amino acid.                                                             <223> OTHER INFORMATION: Description of Artificial - #Sequence: random              sequence.                                                                - - <400> SEQUENCE: 98                                                        - - Xaa Xaa Xaa Xaa Xaa                                                        1               5                                                            - -  - - <210> SEQ ID NO 99                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: histidine           tag sequence.                                                            - - <400> SEQUENCE: 99                                                        - - His His His His His His                                                    1               5                                                            - -  - - <210> SEQ ID NO 100                                                 <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)..(4)                                                      <223> OTHER INFORMATION: The Xaa(s) at postions - #1-3 and 5 can be any             amino acid.                                                             <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (4)                                                           <223> OTHER INFORMATION: The amino acid at post - #ion 4 can be Aspartic            acid or Glutamic acid.                                                  <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic    .                                                                               - - <400> SEQUENCE: 100                                                       - - Xaa Xaa Xaa Asp Xaa                                                        1               5                                                            - -  - - <210> SEQ ID NO 101                                                 <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic      - - <400> SEQUENCE: 101                                                       - - atgggcaaga gaaaaggctc ttaccaagat ctggtggggc ccccttaa  - #                    48                                                                         - -  - - <210> SEQ ID NO 102                                                 <211> LENGTH: 2                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: linker              sequence.                                                                - - <400> SEQUENCE: 102                                                       - - Gly Ser                                                                 __________________________________________________________________________

I claim:
 1. A method for in vitro screening for a transdominantintracellular bioactive agent capable of altering the phenotype of acell, said method comprising the steps:a) introducing a molecularlibrary of randomized candidate nucleic acids into a plurality of cells,wherein each of said nucleic acids comprises a different nucleotidesequence, wherein said randomized candidate nucleic acids are expressedin said cells to produce a plurality of randomized peptides; b)screening said plurality of cells for a cell exhibiting an alteredphenotype, wherein said altered phenotype is due to the presence of atransdominant bioactive agent.
 2. A method according to claim 1 furthercomprising the step:c) isolating said cell exhibiting an alteredphenotype.
 3. A method according to claim 2 further comprising thestep:d) isolating said candidate nucleic acid from said cell.
 4. Amethod according to claim 2 or 3 further comprising the step:e)isolating a target molecule usingi) said candidate nucleic acid; or ii)the expression product of said candidate nucleic acid.
 5. A methodaccording to claim 1 wherein said nucleic acids further comprise apresentation sequence capable of presenting said expression product in aconformationally restricted form.
 6. A method according to claim 1wherein said introducing is with retroviral vectors.
 7. A methodaccording to claim 1 wherein said cells are mammalian cells.
 8. A methodaccording to claim 1 wherein said library comprises at least 10⁴different nucleic acids.
 9. A method according to claim 1 wherein saidlibrary comprises at least 10⁵ different nucleic acids.
 10. A methodaccording to claim 1 wherein said library comprises at least 10⁶different nucleic acids.
 11. A method according to claim 1 wherein saidlibrary comprises at least 10⁷ different nucleic acids.
 12. A methodaccording to claim 1 wherein said library comprises at least 10⁸different nucleic acids.
 13. A method according to claim 1 wherein saidintroducing is with retroviral vectors.
 14. A method according to claim1 wherein each of said candidate nucleic acids is linked to nucleic acidencoding at least one fusion partner.
 15. A method according to claim 14wherein said fusion partner is a presentation sequence capable ofpresenting said expression product in a conformationally restrictedform.
 16. A method according to claim 14 wherein said fusion partner isa rescue sequence.
 17. A method according to claim 14 wherein saidfusion partner is a stability sequence.
 18. A method according to claim14 wherein said fusion partner is a dimerization sequence.
 19. A methodaccording to claim 14 wherein said fusion partner is a targetingsequence.
 20. A method according to claim 19 wherein said targetingsequence is selected from the group consisting of:a) a localizing signalsequence capable of constitutively localizing said translation productto a predetermined subcellular locale; b) a membrane-anchoring sequencecapable of localizing said translation product to a cellular membrane;and c) a secretory signal sequence capable of effecting the secretion ofsaid translation product.
 21. A method according to claim 20 whereinsaid targeting sequence is a nuclear localization signal (NLS).
 22. Amethod according to claim 20 wherein said targeting sequence is amyristylation sequence.
 23. A method for in vitro screening for atransdominant bioactive agent capable of altering the phenotype of acell, said method comprising the steps:a) introducing a molecularlibrary of randomized candidate nucleic acids into a first plurality ofcells, wherein each of said nucleic acids comprises a differentnucleotide sequence; b) contacting said first plurality of cells with asecond plurality of cells; and c) screening said second plurality ofcells for a cell exhibiting an altered phenotype.
 24. A method accordingto claim 23 wherein said randomized candidate nucleic acids areexpressed in said cells to produce a plurality of randomized candidatepeptides.
 25. A method according to claim 24 wherein each of saidcandidate nucleic acids is linked to a nucleic acid encoding at leastone fusion partner.
 26. A method according to claim 25 wherein saidfusion partner is a targeting sequence comprising a secretory signalsequence capable of effecting the secretion of said candidate peptides.27. A method for in vitro screening for a transdominant intracellularbioactive agent capable of altering the phenotype of a cell, said methodcomprising the steps:a) introducing a molecular library of retroviralvectors comprising randomized candidate nucleic acids into a pluralityof cells, wherein each of said nucleic acids comprises a differentnucleotide sequence and wherein said randomized candidate nucleic acidsare expressed in said cells to produce a plurality of randomizedpeptides; b) screening said plurality of cells for a cell exhibiting analtered phenotype, wherein said altered phenotype is due to the presenceof a transdominant bioactive agent.